JP2010523656A - A novel strategy for active substance delivery using micelles and particles - Google Patents

Abstract

The present invention provides biodegradable particles (eg, three-dimensional particles) and micelles that can be used to encapsulate an active agent for delivery to a subject. The present invention further provides methods for generating and delivering such particles and micelles. Furthermore, the present invention provides a vaccination strategy involving the use of these novel particles and micelles. Specifically, the present invention relates to a novel type of hydrophobic polymer containing a ketal group within the polymer backbone. Here, the ketal groups are arranged in such a way that both oxygen atoms are located in the skeleton of the polymer.

Description

  Throughout this application, various publications are cited. The entire disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention belongs.

  This invention was made with government support under NIH / NIAID grants AI048638, AI0556499, AI056947, AI057157, AI0572601, NIH / NIDDK grant DK057665 and Emtech Bio Grant, NIH R01. The government has certain rights in the invention.

(Field of Invention)
The present invention relates to active substances (eg (i) vaccines; (ii) immunomodulators (TLR ligands or synthetic molecules that modulate the function of innate immune cells (eg dendritic cells), or cells (eg dendritic cells) (Including synthetic molecules or siRNAs that modulate signaling networks within or other antigen presenting cells) and / or; (iii) antigen presentation in therapeutic or prophylactic environments to modulate innate and acquired immunity It relates to particles and micelles based on strategies for delivering cells (drugs that target cells).

(Background of the Invention)
Drug delivery vehicles based on polyesters and polyanhydrides have been widely used for sustained release of therapeutic agents due to their excellent biocompatibility profile and slow hydrolysis rate (1). Non-patent document 2; Non-patent document 3; Non-patent document 4). However, many medical applications (eg targeting the acidic environment of lysosomes and tumors) require drug delivery systems that undergo rapid pH-sensitive degradation (Non-Patent Document 5; Non-Patent Document 6). . Most of the degradable polymers used for drug delivery cannot meet this requirement because they are composed of ester bonds that degrade by base-catalyzed hydrolysis at physiological pH values. Particles made from ester-based materials (eg, poly (lactic glycolic acid) (PLGA), polyorthoesters, and polyanhydrides) all produce a large amount of acid upon degradation. This causes degradation of protein and DNA therapeutics, which also takes weeks to months. These materials are not ideal for vaccine development because the life span of mature DC is about 2 days. Recently, pH-sensitive hydrophobic microparticles based on poly (orthoesters) and poly (β-aminoesters) have been successfully used for intracellular drug delivery and tumor targeting, and thus for drug delivery It has shown that it has the possibility of the acid sensitive biomaterial of (nonpatent literature 7; nonpatent literature 8; nonpatent literature 9; nonpatent literature 10). As a result, there is a great deal of interest in developing new strategies for the synthesis of pH sensitive biodegradable polymers.

  Vaccines based on recombinant proteins, peptide antigens, or DNA vaccines encoding such vaccine antigens have great therapeutic potential for infections and tumors in which the antigenic epitopes are defined. Such vaccines had the ability to generate protective immunity against infectious diseases in animal models and in many clinical trials on such vaccines currently under development (Non-Patent Document 11; Non-Patent Document 12; Patent Document 13; Non-Patent Document 14). However, despite their expectations, many of the challenges are the efficiency of peptides, proteins, DNA vaccines and adjuvants that target the appropriate type of antigen-presenting cells to elicit an effective immune response. Concerned about proper delivery. Although promising results have been obtained for peptide vaccines composed of lipid conjugates and PLGA microparticles, there is still a great demand for the development of new peptide vaccine delivery vehicles (Non-Patent Document 15; Non-Patent Document 15). Reference 16).

(Use innate immunity for vaccination)
A characteristic of the immune system is the ability to elicit qualitatively different types of immune responses. Thus, for example, T helper 1 (ie, Th1) immune responses stimulate cytotoxic “killer” T cells that kill virus-infected cells or tumors. Conversely, the T helper 2 (ie, Th2) response is associated with antibody production, particularly secretion of IgE antibodies that confer protection against extracellular parasites or bacteria or toxins. Furthermore, T-regulatory responses can suppress immune responses that are too strong, thus limiting immunopathological symptoms caused by allergies, autoimmunity, graft rejection, or sepsis-like symptoms. The presence of such a broad type of immune response, as well as confer effective protection against viruses, tumors, extracellular parasites and bacteria, and regulate harmful immune responses in allergies, autoimmunity, transplantation and sepsis Taking their unique role into account, modern immunology "Rosetta stones" are learning how to optimally induce an effective immune response in a variety of clinical settings.

  In this sense, recent advances in immunology have revealed the fundamental role of the innate immune system in controlling both the quality and quantity of immune responses (Non-Patent Document 17). Thus, the immune system is non-reactive with most foreign proteins injected in soluble, deaggregated form, but injected with an immunostimulant called an “adjuvant”. It has long been known that these foreign proteins can induce strong immunity. In fact, the essence of an adjuvant is to determine a specific type of subsequent immune response, which can be a cytotoxic T cell response, an antibody response, or a specific class of T helper responses. It was known that this could be biased. (Non-patent document 18; Non-patent document 19; Non-patent document 17). Despite the importance of adjuvants, there is only one alum that has been approved for clinical use in the United States, and most other experimental adjuvants are potent activations of immune cells. It consists of a crude extract of microorganisms or bacteria that induces but also causes toxicity. Until recently, the mechanism of action of such adjuvants was not understood. However, recent advances in innate immunity have provided a conceptual framework for understanding how adjuvants work. The core of this problem is a rare but widely distributed network of cells known as dendritic cells (DCs), which constitutes an essential component of the innate immune system. DCs called “natural adjuvants” express receptors that can recognize microbial and viral components. Such receptors include Toll-like receptors (TLRs), C-type lectins, and caterpillar proteins that can "sense" microbial stimuli and activate DCs and other immune cells. Non-patent document 18; Non-patent document 19; Non-patent document 17). It is now clear that DCs play an essential role in regulating the quality and quantity of immune responses.

  Currently, there are about 13 TLRs described for mammals. Activating different TLRs on DCs induces qualitatively different types of immune responses (Pulendran et al., 2001 supra; Dillon et al., 2004 supra; Non-patent document 20; Non-patent document 21). Thus, activating most TLRs can induce a Th1 response; activating TLR3, 7 or 9 can induce virally infected cells and cytotoxic T cells that kill tumors; and The evidence obtained shows that activating TLR2 suppresses a Th2 response (associated with an antibody response that provides protection against viruses or extracellular bacteria or parasites), or an immune response that is too strong, and thus Suggests that it also induces T-regulatory or tolerogenic responses (providing protection against allergy, autoimmunity, sepsis, and uncontrolled immunity in transplantation). Thus, DC and TLR and other recognition receptors represent attractive immunomodulating targets for vaccinologists and drug developers. Therefore, learning how to develop the basic elements of the innate immune system (eg, DC and TLR) is of paramount importance in the development of new drugs and vaccines.

  An important conclusion derived from this concept is that the vast majority of vaccines that have been developed over the past 200 years (since the first recorded Edward Jenner vaccination trial) have been experimentally developed. . Thus, despite vaccine success in controlling various calamities (eg smallpox, polio, TB and yellow fever), how do these vaccines stimulate such effective immunity? I have no knowledge of scientific reasons for this. For example, yellow fever vaccine 17D [YF-17D] is one of the most effective known vaccines. Since the development of yellow fever vaccine 17D before 65 years, this vaccine has been administered to over 400 million people worldwide. Despite its success, its mechanism of action is unknown. Thus, as noted above, the striking advances in innate immunity that have occurred over the past six years have allowed us to devise future vaccines against emerging and re-emerging infections in the 21st century. Provides a new view that can be used to understand how such a “gold standard” vaccine works. In this sense, our recent finding is that a very effective yellow fever vaccine (YF-17D) is a potent stimulator of DC and many TLRs (including TLR2, 7, 8, and 9) (Non-Patent Document 22). Taking into account the different types of immune responses caused by different TLRs (Pulendran et al., 2001; Non-Patent Document 20; Dillon et al., 2004; Non-Patent Document 21), a number of TLRs are taken into account. It was attractive to speculate that YF-17D induces a broad spectrum of immune responses by activating. In fact, our data show that YF-17D causes a broad spectrum of innate and acquired immune responses (Th1 cells, Th2 cells, cytotoxic T cells, neutralizing antibodies) and different TLRs have different types of this This suggests that multivalent immunity is controlled (Non-patent Document 22). Inducing such a broad spectrum of immune responses is also a vaccine against other infectious diseases (eg, HIV, HCV, malaria, TB, influenza, charcoal destruction and Ebola) for which there is currently no effective vaccine, or against tumors May be advantageous for designing. Thus, strategies for designing future vaccines against emerging or re-emerging infections may benefit from incorporating multiple TLR ligands and antigens and immunomodulators to induce a wide variety of immune responses . Thus, an important challenge is the development of delivery systems that have the ability to deliver such immunomodulators in vivo.

  Congestive heart failure is a leading cause of morbidity and mortality worldwide and there is a great need for effective treatment options. Cardiac dysfunction after myocardial infarction is a progressive disease, and any successful treatment is required over the course of days / weeks (Non-Patent Document 23).

  Patients with acute myocardial infarction are traditionally treated in hospitals by angioplasty that attempts to remove blood diluent and / or affected vessels. Local cell death that occurs during this acute phase results in chronic heart failure characterized by increased ventricular size and reduced contractile function (23). Currently, the only treatment for heart failure is transplant surgery, and it is estimated that less than 30% of transplant patients receive a new heart and survive (Rosamond et al., Heart Disease and Stroke Statistics- 2007 Update, A Report From the American Heart Association Statistics Committee and Stroke Statistics Subcommittee, Circulation, 2006). Many methods performed immediately after the infarction are the targets of potential therapeutic approaches.

  Myocardial loss is mainly localized, suggesting that localized treatment is most promising (Non-Patent Document 23). In recent years, many clinical studies have begun to deliver localized treatments in the form of various cell types for myocardial remodeling (Assmus et al., Transplantation of Progenitor Cells and Generation Enhancement in Myocardial Infection (Assmus et al. TOPCARE-AMI) Circulation, 2002. 106 (24): 3009-17, Kang et al., Effects of intracoronary infusion of peripheral blood-mobile mobilized witness. cular systolic function and restenosis after coronary stenting in myocardial infarction:. the MAGIC cell randomised clinical trial Lancet, 2004. 363 (9411): 751-6).

  Due to the complex nature of direct protein, RNA and DNA injections, efforts are currently focused on using biomaterials to deliver therapeutic agents to the myocardium (Davis et al., Custom design of the. cardiac microenvironment with biomaterials. Circ Res, 2005. 97 (1): 8-15). Although many studies still utilize intravenous injections or oral treatments, the exact mechanism of action remains relatively unknown. Specifically, it has not been investigated how these molecules / proteins can enter orally or intravenously and exert significant effects only on the heart. Our previous work has used direct intramyocardial injection for the delivery of biomaterials that carry proteins and drugs (Davis et al., Local myocardial insulin-like growth factor 1 (IGF- . 1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction Proc Natl Acad Sci U S A, 2006. 103 (21): 8155-60, Davis et al., Injectable self-assembling peptide nanofibers create intramyocardial microenv irrationals for endothelial cells. Circulation, 2005. 111 (4): 442-50, Hsieh et al., Controlled delivery of PD-B for mycardial protection using PD. -48). Some consider this harmful, but it is becoming more and more common for human use. This is because several companies have developed catheters that can deliver cells and drugs from the interior of the chamber to the myocardial wall. Furthermore, using a novel technique, researchers were able to deliver microspheres to the myocardial wall of mice in a closed-chest procedure (Springer et al., Closed-chest cell injections into mouse myocardium). guided by high-resolution echography. Am J Physiol Heart Circ Physiol, 2005. 289 (3): H1307-14). Accordingly, there is great interest in developing small, injectable particles for myocardial therapy.

p38 MAP kinase Certain inflammatory cytokines are significantly increased in the post-infarction myocardium (Bolli, R., Oxygen-derived free radicals and myocardial reperfusion in: an overview. 19 pour. : 249-68, Bolli et al., Direct evidence that oxygen-derived free radicals contribute to postsistic mycological dysfunction in the intact. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression Circ Res, 2004. 94 (4): 514-24). These cytokines activate specific signaling pathways that lead to the death of adult cardiomyocytes and local stem cell populations.

  Three different mitogen-activated protein kinase (MAPK) cascades have been identified and studied as cell signaling pathways. The p38 MAPK cascade is well characterized in stress and inflammatory responses (Lai et al., The role of MAP kinases in trauma and ischemia-reperfusion. J Invest Surg, 2004. 17 (1): 45-53, Lopez-Neplice- And Toledo-Pereyra, Phosphoration of signal transduction pathways in ischemia and reperfusion J Surge Res, 2006. 134 (2): 292-9). In addition, through the use of transgenic mouse models and gene therapy studies, the p38 MAPK pathway has been implicated in the death of adult cardiomyocytes (Engel et al., P38 MAP kinase inhibition of adult mammary cardiology. 19 (10): 1175-87, Awad et al., Obese diabetic mouse environmental differential effects primitive and monofunctional endogenous cell progenitors. 5) (5).

  There are four different isoforms of p38, and p38α is the most studied and involved in the disease process. Like several MAPKs, p38 is activated by dual phosphorylation (using ATP as a phosphate donor) via various upstream mechanisms, and its function is conserved across several cell types. Yes. Activation of p38 in macrophages results in increased superoxide production and pro-inflammatory cytokine expression. In fibroblasts, activation of p38 results in the production of cytokines and activation of the pro-fibrotic cascade (Kumar et al., P38 MAP kinases: key signaling molecules as therapeutic targets. Drug Discov, 2003. 2 (9): 717-26). Finally, in cardiomyocytes, activation of p38 leads to apoptosis and cytokine release (Li et al., Selective inhibition of p38 alpha MAPi im ri ri ri di c h a ly pi m imo ri pi a ri m i s s s s s s s s s s s s s s e m e m e n e i n e n i n e r i n e n i n e n i n e n i n e n i n e n i n e n i n i n e n i n i n e n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n. 2006. 291 (4): H1972-7, Ren et al., Role of p38 alpha MAPK in cardiac apoptosis and remodeling after myocardial infusion J Mol Cell Cardol, 2005. 38 (4): 617-230). All three of these processes play an important role in the progression of cardiac dysfunction after infarction. FIG. 29 shows a schematic diagram of p38 activation, including upstream effector activation and activation results.

  Phosphorylation of p38 leads to the activation of two important pathways in the course of the disease, including caspases and nuclear factor κB (NFκB) (Chen and Tu, Apoptosis and heart failure: mechanisms and therapeutic Amplications. Drugs, 2002. 2 (1): 43-57). During apoptosis, phosphorylation of p38 induces Bax activation, which ultimately leads to mitochondrial permeability, leading to caspase cleavage and induction of apoptosis. In an alternative pathway after p38 phosphorylation, the inhibitory protein IκB is phosphorylated, releasing NFκB into the nucleus where activation of inflammatory cytokines can be initiated (Agarwal et al., TNF blockade: an inflammatory issue. Ernst Schering Res Found Workshop, 2006 (56): 161-86). Both of these pathways have been targeted for a variety of inflammatory diseases including arthritis, ischemia / reperfusion injury and airway inflammation (Herala and Brown, p38 MAPK signaling cascades in inflammatory disease. Mol Med Today. , 1999. 5 (10): 439-47, Karin, M., Inflammation-activated protein kinases as targets for drug development. Proc Am Torac Soc, 2005. 2 (4): 386 sc; Inhibition of p38 MAP Kinase as a therapeut i pharmacology, 2000. 47 (2-3): 185-201, Peifer et al., New approaches to the treatment of the infrastructure disorders, and the cult of humans. -49, Schiven, GL, The biology of p38 kinase: a central role in information Curr Top Med Chem, 2005. 5 (10): 921-8).

  Inhibition of p38 activation by genetic knockout model, gene therapy and inhibitor administration had a beneficial effect in preventing systolic dysfunction after myocardial infarction (Engel et al., P38 MAP kinase inhibition of adult mammalian cardiology) Genes Dev, 2005. 19 (10): 1175-87, Minamino et al., MEKK1 suppressed oxidative stress-in-the-op-of-a-brid. :. 15127-32, Kaiser et al., Inhibition of p38 reduces myocardial infarction injury in the mouse but not pig after ischemia-reperfusion Am J Physiol Heart Circ Physiol, 2005. 289 (6): H2747-51, Kumar et al., P38 MAP kinases : Key signaling molecules as therapeutic targets for inflammatory diseases.Nat Rev Drug Discovery, 2003.2 (9): 717-26, Kulik et al., Antioptopot. ng by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt Mol Cell Biol, 1997 17 (3):. 1595-606, Liu et al., Inhibition of p38 mitogen-activated protein kinase protects the heart against cardiac remodeling in rice with heart failure reselling from mycardial information J Card Fail, 2005. 11 (1): 74-81, See et al., P38 mitogen-activated protein kinase inhibition improves cardiac function and attentuates left ventilatory remodeling flooding. J Am Coll Cardiol, 2004. 44 (8): 1679-89). This is widely believed to be due to a variety of mechanisms including inhibition of apoptosis, induction of cardiomyocyte proliferation, reduction of oxidative stress, and prevention of expression of pro-fibrotic genes. When delivered to mice, the p38 inhibitor, SB-282, improved cardiac function in a rat model of cardiac injury (Li et al. J Physiol Heart Circ Physiol, 2006. 291 (4): H1972-7). Rats fed this compound for several days after L-NAME, salt and angiotensin II treatment showed significant improvements in systolic function and other cardiac parameters. Furthermore, in a mouse model of ischemia / reperfusion, SB239063 injected via the tail vein immediately before ischemia prevented p38 phosphorylation and reduced infarct size 24 hours later. It was noted in this study that this finding was not transferred to larger animals. Pigs were subjected to a similar procedure but showed no benefit of p38 inhibition immediately after reperfusion. In this study, however, it is important to understand that researchers injected pigs prior to ischemia-reperfusion to give the left ventricular lumen an inhibitor as a bolus dose (Kaiser et al., Inhibition). of p38 reduced mycardial information injury in the mouse but not pig after ischemia- reperfusion Am J Physiol Heart Circ. Thus, it is unlikely that much of the inhibitor was retained in the myocardium in the required time frame. Interestingly, recent studies performed using the p38 inhibitor SB203580 demonstrated an important role for p38 inhibition in regulating the ability of adult cardiomyocytes to reenter the cell cycle. Treatment with a p38 inhibitor every 3 days resulted in a significant increase in staining of myocardial cell proliferation markers after myocardial infarction (Engel et al., P38 MAP kinase inhibition of adult mammalian cardiomy. 200 ed. ): 1175-87). Interestingly, the exact benefit is unclear, as is the total amount of circulating myocytes rising by less than 1%. Our proposals investigate the effect of sustained p38 inhibition on apoptosis of larger rodent cardiomyocytes and create a means to investigate the sustained release of p38 inhibitors in larger mammals.

  Although the p38 kinase inhibitor SB239063 prevented cardiac dysfunction in mice after infarction, this effect was not transferred to larger animals (Burnham et al. Am J Respir Crit Care Med, 2005. 172 (7): 854-60). This is most likely due to the small size of the inhibitor and its tendency to diffuse quickly from the injection site. The most successful therapeutic outcomes using small molecule inhibitors require multiple injections over a longer period to restore function, which is impractical for human intervention. For these reasons, therapeutic approaches that require only a single administration / injection are highly advantageous over existing treatment options.

  As described herein, polyketal (PK) particles are a new class of biomaterials that hydrolyze in a controllable manner at physiological pH values and break down into neutral compounds.

Anderson, J.M. M.M. Et al., Adv. Drug Delivery Rev. 1997, 28, p. 5-24 Jain, R.A. A. , Biomaterials, 2000, 21, p. 2475-2490 Mathiowitz, E .; J. et al. Appl. Polym. ScL, 1988, 35, p. 755-774 Berkland, C.I. J. et al. Controlled Release, 2004, 94, p. 129-141 Stubbs, M.M. Et al., Mol. Med. Today, 2000, 6, p. 15-19 Leroux, J. et al. -C. , Adv. Drug Delivery Rev. 2004, 56, p. 925-926 Heller, J .; Et al., Biomacromolecules, 2004, 5, p. 1625-1632 Heller, J .; Et al., Adv. Drug Delivery Rev. , 2002, 54, p. 1015-1039 Berry, D.C. Chem. Biol, 2004, 11, p. 487-498 Potinini, A .; J. et al. Controlled Release, 2003, 86, p. 223-234 van Endert, PM, Biologicals, 2001, 29, p. 285-8 Purcell, AW et al., Journal of Peptide Science, 2003, 9, p. 255-81 Shirai, M .; Et al., Journal of Virology, 1994, 68, p. 3334-42 Hunziker, IP et al., International Immunology, 2002, 14, p. 615-26 Ertl, HCJ et al., Vaccine, 1996, 14, p. 879-85 Jackson, DC et al., Vaccine, 1997, 15, p. 1697-705 Pulendraun & Ahmed, Cell, 2006, 124, p. 849-863 Pulendran, Immunol. Rev. 2004, 199, p. 227-250 Pulendran, J. et al. Immunol. 2005, 175, p. 2457-2465 Agrawal et al. Immunol. , 2003, 171, p. 4984-4899 Dillon et al. Clin. Immunol, 2006, 116, p. 916-928 Querec et al., J. MoI. Exp. Med. 2006, 203, p. 413-421 Anversa, P.A. , "Myocycle death in the pathological heart." Circ Res, 2000, 86 (2) p. 121-4

(Summary of the Invention)
The present invention provides biodegradable particles (eg, three-dimensional particles) and micelles that can be used to encapsulate an active agent for delivery to a subject. The present invention further provides methods for producing and delivering such particles and micelles. Furthermore, the present invention provides a vaccination strategy involving the use of these novel particles and micelles.

(Hydrophobic polyketal particles)
The present invention is directed to a novel type of hydrophobic polymer containing a ketal group within the polymer backbone. Here, the ketal groups are arranged in such a way that both oxygen atoms are located in the skeleton of the polymer.

  Furthermore, the ketal polymer can be formed by a ketal exchange reaction between the ketal and the diol. In accordance with the present invention, one or more types of ketals and / or diols can be used for the formation of homopolymers or copolymers.

  Also included by the present invention are polyketal polymers linked by other polymers (eg, PEG, polyester, polyamide, polysaccharide, polyether, or polyanhydride). The resulting polymer can be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. Polythioketal polymers mixed with polythio-amine ketals, polythio-hydroxyl ketals and polyhydroxyl-amine ketals are also within the scope of the present invention.

  The polyketal polymer of the present invention is hydrolyzed in an aqueous solution into low molecular weight, water soluble alcohols and ketones. The advantage of a ketal bond within the backbone is that this ketal bond degrades at pH 5.0 within 1-2 days under the phagosomal acidic conditions. Thus polyketals can also be used to target the acidic environment of tumors, inflammation and phagolysosomes. This degradation does not produce acidic degradation products. Therefore, the ketal polymer is suitable for biological use.

(Micelle)
The present invention further provides novel biodegradable cross-linked micelles containing multiple polymers. Here, the polymer is crosslinked by, for example, an external crosslinking agent (that is, a drug that is not introduced into the polymer chain). The advantage of using an external agent is that the cross-linking reaction is faster compared to reactions in which only cross-linkable moieties within the polymer are used. The external cross-linking agent also reduces the chance that the encapsulated protein is destroyed.

FIG. 1 is a bar graph showing the fluorescence intensity of filtered FITC-Ova-containing PKN and unfiltered FITC-Ova-containing PKN (excitation 494 nm, emission 520 nm). The data shown is the average of two samples. FITC-Ova encapsulation efficiency is 60% (Example 3, below). FIG. 2 is a schematic view showing synthesis and decomposition of a ketal skeleton polymer (polyketal). (A) Ketal exchange reaction between 1,4-benzenedimethanol and 2,2-dimethoxypropane to produce ketal intermediate 1. (B) One step polymerization to produce polyketal 2. Reaction steps A and B proceed by distilling the by-product methanol. (C) Formation of drug loaded particles by solvent evaporation method. The particles are pH sensitive and break down into small molecule releasable compounds (Example 4, below). FIG. 3 (A) is a graph showing a GPC trace (Shimadzu SCL-10A) of polyketal 2 (of FIG. 2) in THF. Polystyrene standards _{(Polymer Laboratories, Inc.) M} w = 4000, M W / M n = 1.54 , based on the. The Y-axis shows the relative absorbance at 262 nm. FIG. 3 (B) shows the ¹ H NMR spectrum of polyketal 2 (of FIG. 2) in CDCI ₃ (Varian Mercury Vx 400); the repeat unit peaks are 7.3 ppm (4b), 4.5 ppm ( 4c) and 1.5 ppm (6a). The peaks at 2.5 and 1.0 are attributed to triethylamine added to prevent ketal hydrolysis (Example 4, below). FIG. 4 is a line graph showing the hydrolysis kinetics of polyketal 2 (of finely ground powder) at pH 1.0, 5.0 and 7.4 (of FIG. 2). The half-life of exponential decay is 102 hours (pH 7.4) and 35 hours (pH 5.0). The pH 1.0 control batch was fully hydrolyzed before the first time point (Example 4, below). FIG. 5 shows an SEM image of particles made of polyketal 2 (of FIG. 2). (A, B) Particles using a ratio of PVA to Polyketal 2 of 0.2: 1 (particle size: 0.5-30 μm). (C) Particles loaded with dexamethasone (particle size: 200-500 nm) made using 1: 1 PVA: Polyketal 2. The scale bars are (A) 80 μm, (B) 3 μm, and (C) 4 μm (Examples 5 and 6, below). FIG. 6 is a schematic diagram showing particle formation. A. Step 1: Dissolve polyketal and drug in chloroform; dissolve polyvinyl alcohol in water. B. Step 2: Add chloroform solution to water and sonicate to produce micron sized droplets. Step 3: Evaporate chloroform to produce particles (Example 6, below). FIG. 7 is a photograph showing that polyketal particles loaded with fluorescein are taken into the liver. Mouse liver tissue section showing release of fluorescein from PKN after intravenous injection (Example 6, below). FIG. 8 is a schematic showing the design and synthesis of peptide-crosslinked micelles. Step 1: ISS DNA and I are mixed to form micelles (non-crosslinked micelles). Step 2: These micelles are then cross-linked with antigenic peptide (II) to produce a delivery system that can encapsulate both immunostimulatory molecules and peptide antigens. After phagocytosis by APC, peptide-crosslinked micelles release their constituents (Example 6, below). FIG. 9 shows the synthesis and characterization of PEG-polylysine thiopyridal. A is a chemical scheme showing the synthesis of PEG-polylysine thiopyridal. B is a ¹ H-NMR spectrum of PEG-PLL-thiopyridal in D ₂ O. C / D is a graph showing dynamic light scattering analysis of uncrosslinked PCM (C) and peptide crosslinked (D). E is a graph showing the crosslinking reaction of cysteine in peptide antigen II (FIG. 8). F is a graph showing a UV analysis of the cross-linking reaction between peptide antigen II (FIG. 8) and block copolymer micelle (Example 6, below). FIG. 9 shows the synthesis and characterization of PEG-polylysine thiopyridal. A is a chemical scheme showing the synthesis of PEG-polylysine thiopyridal. B is a ¹ H-NMR spectrum of PEG-PLL-thiopyridal in D ₂ O. C / D is a graph showing dynamic light scattering analysis of uncrosslinked PCM (C) and peptide crosslinked (D). E is a graph showing the crosslinking reaction of cysteine in peptide antigen II (FIG. 8). F is a graph showing a UV analysis of the cross-linking reaction between peptide antigen II (FIG. 8) and block copolymer micelle (Example 6, below). FIG. 9 shows the synthesis and characterization of PEG-polylysine thiopyridal. A is a chemical scheme showing the synthesis of PEG-polylysine thiopyridal. B is a ¹ H-NMR spectrum of PEG-PLL-thiopyridal in D ₂ O. C / D is a graph showing dynamic light scattering analysis of uncrosslinked PCM (C) and peptide crosslinked (D). E is a graph showing the crosslinking reaction of cysteine in peptide antigen II (FIG. 8). F is a graph showing a UV analysis of the cross-linking reaction between peptide antigen II (FIG. 8) and block copolymer micelle (Example 6, below). FIG. 9 shows the synthesis and characterization of PEG-polylysine thiopyridal. A is a chemical scheme showing the synthesis of PEG-polylysine thiopyridal. B is a ¹ H-NMR spectrum of PEG-PLL-thiopyridal in D ₂ O. C / D is a graph showing dynamic light scattering analysis of uncrosslinked PCM (C) and peptide crosslinked (D). E is a graph showing the crosslinking reaction of cysteine in peptide antigen II (FIG. 8). F is a graph showing a UV analysis of the cross-linking reaction between peptide antigen II (FIG. 8) and block copolymer micelle (Example 6, below). FIG. 10 shows the effect of GSH on peptide and DNA release. A is a graph showing GSH sensitive peptide release. B is a gel electrophoresis analysis showing GSH sensitive DNA release. C is a gel electrophoresis analysis showing that ISS-DNA is protected from serum nucleases in PCM (Example 6, below). FIG. 11 represents a block copolymer micelle. A shows the chemical structure of PEG-poly (lysine-thio-pyridyl). B is a schematic diagram showing micelle formation. C is a schematic view showing micelle cross-linking. D is a schematic showing the reduction of cross-linked micelles (Example 6, below). FIG. 11 represents a block copolymer micelle. A shows the chemical structure of PEG-poly (lysine-thio-pyridyl). B is a schematic diagram showing micelle formation. C is a schematic view showing micelle cross-linking. D is a schematic showing the reduction of cross-linked micelles (Example 6, below). FIG. 12 shows micelle immunology. A. Confocal microscopic analysis of uptake of SIINFEKL-CFSE micelles by DC-derived human monocytes. B. FACS analysis of uptake of SIINFEKL peptide encapsulated micelles by DC-derived human monocytes. C. FACS analysis of uptake of SIINFEKL peptide encapsulated micelles by mouse DCs and macrophages (Example 6, below). FIG. 12 shows micelle immunology. A. Confocal microscopic analysis of uptake of SIINFEKL-CFSE micelles by DC-derived human monocytes. B. FACS analysis of uptake of SIINFEKL peptide encapsulated micelles by DC-derived human monocytes. C. FACS analysis of uptake of SIINFEKL peptide encapsulated micelles by mouse DCs and macrophages (Example 6, below). FIG. 12 shows micelle immunology. A. Confocal microscopic analysis of uptake of SIINFEKL-CFSE micelles by DC-derived human monocytes. B. FACS analysis of uptake of SIINFEKL peptide encapsulated micelles by DC-derived human monocytes. C. FACS analysis of uptake of SIINFEKL peptide encapsulated micelles by mouse DCs and macrophages (Example 6, below). FIG. 13 is a bar graph showing that micelles formulated with SIINFEKL peptide induce a strong T cell response in vitro (Example 6, below). FIG. 14 shows micelle immunology. A shows FACS analysis for the efficient uptake of micelles encapsulated OVA protein by mouse DCs and macrophages. B is a graph showing that micelles encapsulating OVA / CpG activate DC in vitro (Example 6, below). FIG. 14 shows micelle immunology. A shows FACS analysis for the efficient uptake of micelles encapsulated OVA protein by mouse DCs and macrophages. B is a graph showing that micelles encapsulating OVA / CpG activate DC in vitro (Example 6, below). FIG. 15 is a graph showing the immunology of polyketal particles. Uptake of U0126 encapsulated polyketal particles (PKN) by mouse DCs and macrophages in vitro (Example 6, below). FIG. 16 is a schematic diagram showing an outline of an experiment on stimulation of T cells in vitro using an OVA-OT / 1 transgenic model (Example 6, below). FIG. 17 shows micelle immunology. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that spleen cells pulsed with antigen formulated micelles induce strong antigen-specific CD8 + T cell responses in vitro. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that DCs pulsed with antigen formulated micelles induce strong antigen-specific CD8 + T cell responses in vitro. C. Flow cytometry showing that micelles formulated with antigen activate DCs in vivo (Example 6, below). FIG. 17 shows micelle immunology. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that spleen cells pulsed with antigen formulated micelles induce strong antigen-specific CD8 + T cell responses in vitro. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that DCs pulsed with antigen formulated micelles induce strong antigen-specific CD8 + T cell responses in vitro. C. Flow cytometry showing that micelles formulated with antigen activate DCs in vivo (Example 6, below). FIG. 17 shows micelle immunology. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that spleen cells pulsed with antigen formulated micelles induce strong antigen-specific CD8 + T cell responses in vitro. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that DCs pulsed with antigen formulated micelles induce strong antigen-specific CD8 + T cell responses in vitro. C. Flow cytometry showing that micelles formulated with antigen activate DCs in vivo (Example 6, below). FIG. 18 shows the immunology of micelles and polyketal particles. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 + IFN-γ + T cell response in vivo. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 ⁺ TNFα ⁺ T cell response in vivo. C. Line graph showing kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA / CpG vaccination. D. Line graph showing the kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA + UOl26 PKN vaccination. E. Bar graph showing that micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. F. Bar graph showing vaccine-formulated micelles induce antigen-specific IgE and IgM antibody responses in vivo (Example 6, below). FIG. 18 shows the immunology of micelles and polyketal particles. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 + IFN-γ + T cell response in vivo. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 ⁺ TNFα ⁺ T cell response in vivo. C. Line graph showing kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA / CpG vaccination. D. Line graph showing the kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA + UOl26 PKN vaccination. E. Bar graph showing that micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. F. Bar graph showing vaccine-formulated micelles induce antigen-specific IgE and IgM antibody responses in vivo (Example 6, below). FIG. 18 shows the immunology of micelles and polyketal particles. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 + IFN-γ + T cell response in vivo. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 ⁺ TNFα ⁺ T cell response in vivo. C. Line graph showing kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA / CpG vaccination. D. Line graph showing the kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA + UOl26 PKN vaccination. E. Bar graph showing that micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. F. Bar graph showing vaccine-formulated micelles induce antigen-specific IgE and IgM antibody responses in vivo (Example 6, below). FIG. 18 shows the immunology of micelles and polyketal particles. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 + IFN-γ + T cell response in vivo. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 ⁺ TNFα ⁺ T cell response in vivo. C. Line graph showing kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA / CpG vaccination. D. Line graph showing the kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA + UOl26 PKN vaccination. E. Bar graph showing that micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. F. Bar graph showing vaccine-formulated micelles induce antigen-specific IgE and IgM antibody responses in vivo (Example 6, below). FIG. 18 shows the immunology of micelles and polyketal particles. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 + IFN-γ + T cell response in vivo. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 ⁺ TNFα ⁺ T cell response in vivo. C. Line graph showing kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA / CpG vaccination. D. Line graph showing the kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA + UOl26 PKN vaccination. E. Bar graph showing that micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. F. Bar graph showing vaccine-formulated micelles induce antigen-specific IgE and IgM antibody responses in vivo (Example 6, below). FIG. 18 shows the immunology of micelles and polyketal particles. A. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 + IFN-γ + T cell response in vivo. B. The left panel is a flow cytometric analysis and the right panel is a summary showing that vaccine-formulated micelles induce a strong antigen-specific CD8 ⁺ TNFα ⁺ T cell response in vivo. C. Line graph showing kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA / CpG vaccination. D. Line graph showing the kinetics of specific CD8 ⁺ / IFNγ ⁺ T cells after OVA + UOl26 PKN vaccination. E. Bar graph showing that micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. F. Bar graph showing vaccine-formulated micelles induce antigen-specific IgE and IgM antibody responses in vivo (Example 6, below). FIG. 19 shows a polyketal derived from cyclohexanedimethanol (referred to herein as PCADK and having the IUPAC name poly (cyclohexane-1,4-diylacetone dimethylene ketal)). A. Chemical notation indicating a polyketal derived from cyclohexanedimethanol. B. Line graph showing that PCADK degrades in an acid sensitive manner (Example 6, below). FIG. 20 shows an SEM image showing that PCADK-derived particles can encapsulate hydrophobic compounds and drugs (eg, rhodamine red and ebselen) (Example 6, below). FIG. 21 shows a line graph showing that the release of rhodamine red from PCADK is pH sensitive (Example 6, below). FIG. 22 is a chemical notation showing that polyketals containing almost any aliphatic diol can be made (Example 6, below). FIG. 23 is a photograph showing that FITC-labeled polyketals are phagocytosed by liver macrophages (Example 6, below). FIG. 24A is a schematic showing the procedure of a double emulsion used to encapsulate catalase and superoxide dismutase in polyketal particles. B is an SEM image of catalase-containing particles and a fluorescence microscope image of catalase-containing particles. C is a graph showing that catalase particles have enzyme activity (Example 6, below). FIG. 24A is a schematic showing the procedure of a double emulsion used to encapsulate catalase and superoxide dismutase in polyketal particles. B is an SEM image of catalase-containing particles and a fluorescence microscope image of catalase-containing particles. C is a graph showing that catalase particles have enzyme activity (Example 6, below). FIG. 24A is a schematic showing the procedure of a double emulsion used to encapsulate catalase and superoxide dismutase in polyketal particles. B is an SEM image of catalase-containing particles and a fluorescence microscope image of catalase-containing particles. C is a graph showing that catalase particles have enzyme activity (Example 6, below). FIG. 25 is a chemical notation showing a polyketal made by acyclic diene metathesis (ADMET). FIG. 26 is a schematic diagram for synthesis and acid degradation of drug loaded particles. FIG. 27 is a table showing the conditions used to make the rhodamine-containing PCADK particles different. FIG. 28 is a graph showing that His-GFP bound stably to NTA-Ni for at least 15 hours (Example 9, below). FIG. 29 is a schematic showing the source of p38 activation and downstream effects (Example 7, below). FIG. 30 is a schematic diagram of the synthesis of poly (1,4-cyclohexane-acetone dimethylene ketal) (PCADK). PCADK is synthesized using an acetal exchange reaction using 1,4-cyclohexanedimethanol and 2,2 dimethoxypropane as reactants. PCADK hydrolyzes to 1,4-cyclohexanedimethanol and acetone (Example 7, below). FIG. 31 is a graph showing a GPC trace of PCADK in THF. The Y-axis shows the relative UV absorbance at 262 nm. Mw = 6,282, polydispersity index (PDI) = 1.54 (Example 7, below). FIG. 32 is a photograph showing a representative SEM image of particles (PK-p38 _i ) loaded with SB239063. These images reflect a particle size of approximately 3-15 μm (return) (Example 7, below). FIG. 33 is a photograph showing that the particle size can be easily modified. A) SEM of particles produced by the original protocol. B) Higher magnification SEM of particles produced from reduced homogenization rate. C) High magnification image of particles generated from reduced PVA concentration (Example 7, below). FIG. 34 is a photograph showing a representative SEM image of an alternative porous particle. Addition of N-hexane to the initial dispersion resulted in the formation of porous particles. The approximate size of these particles is 10-25 μm (Example 7, below). FIG. 35 is a photograph showing an SEM image of SOD-PKN. (A) 6000 × magnification. (B) 1000 × magnification (Example 7, below). FIG. 36 is a chart showing grouped data from cultured macrophages pretreated with PK-SOD and stimulated with LPS. Pretreatment of macrophages with polyketals loaded with SOD resulted in a significant reduction in LPS-stimulated superoxide release compared to empty PK and free SOD (Example 7, below). FIG. 37 is a photograph showing a representative fluorescence image of a polyketal loaded with FITC incubated with cultured macrophages. Macrophages were incubated with PK-FITC for 2 hours, followed by thorough washing and imaging. Arrows indicate cells that have taken up FITC dye. It can also be seen that empty particles are bound to the surface of the macrophages (Example 7, below). FIG. 38 shows representative Western blot (top) and grouped concentration data from macrophages pre-treated with polyketals, stimulated with 10 ng / mL TNF-α for 20 minutes as indicated (PK time). (Densitometric data) (bottom). Macrophages were incubated with empty PK or PK-p38 for 2-6 hours, followed by extensive washing and TNF-α stimulation. Longer pretreatment with empty PK did not alter p38 phosphorylation, but there was significant inhibition of p38 phosphorylation with 4 and 6 h PK-p38 _i pretreatment (* p <0.05; ANOVA according to Tukey-Kramer; n = 4) (Example 7, below). FIG. 39 is a chart showing grouped data from macrophages pretreated with polyketals for 6 hours and stimulated with 10 ng / mL TNF-α for 20 minutes and dihydroethidium for 20 minutes. HPLC data showing a significant increase in extracellular superoxide release by TNF-a treatment. This increase was not disturbed by empty PK but was completely inhibited by PK-p38 _i pretreatment (* P <0.05 ANOVA according to Tukey-Kramer (Example 7, below)). FIG. 40 shows detection of extracellular superoxide production after infarction by DHE-HPLC. The top panel is a representative trace from sham and infarct (MI) samples. The bottom panel is normalized data showing the increase in extracellular superoxide production detected by this method. The animals were subjected to sham surgery or coronary ligation surgery and the left ventricular free wall was collected 3 days after the infarction. Fragments were incubated with dihydroethidium in the presence and absence of exogenous SOD and data expressed as oxy-ethidium concentration inhibited by SOD (n = 4). These data represent potential mechanisms of post-infarction damage that can be examined in future experiments (Example 7, below). FIG. 41 is a photograph showing representative H & E staining of leg muscle 3 days after infarction. In the left panel, injected PCADK forms a defined injection area with slight cell staining outside the border. In contrast, PLGA (right) shows evidence of a population of fibrosis and inflammatory cells (Example 7, below). FIG. 42 is a photograph showing that polyketal injection causes slight inflammation. The left panel shows two representative immunofluorescence stains for CD45 (a common inflammatory marker). There was little staining in the sections. In contrast, on the right, leg muscles injected with PLGA (a commonly used polymer) produce a robust inflammatory response as measured by CD45 staining (Example 7, below). FIG. 43 shows that PK-p38 _i treatment inhibits post-infarct p38 phosphorylation. Representative Western blot for both phospho-p38 and total p38 in the infarct zone 3 days after infarction. Grouped data (mean + SEM) demonstrate a significant increase in phosphorylation of p38 in the MI and MI + PK groups (* p <0.05 vs sham and MI + PK-p38 _i ; n = 13 total mice) (Example 7, below). FIG. 44 shows that PK-p38 _i treatment inhibits IL-5 expression after infarction. Representative Western blot for both IL-5 and β-actin in the infarct zone 3 days after infarction. Grouped data demonstrates a significant increase in normalized IL-5 levels in the MI and MI + PK groups (* p <0.05 vs sham and MI + PK-p38 _i ). Data are expressed as mean + SEM (n = 13 total mice) (Example 7, below). FIG. 45 is a chart showing that SB239063 encapsulated with polyketal inhibits TNF-α production 3 days after infarction. Grouped data demonstrating that a single injection of PK-p38 _i almost normalizes TNF-α levels in post-infarction rats (n = 19 total rats). Data are expressed as mean + SEM (Example 7, below). FIG. 46 is a chart showing that killing of transplanted cells was reversed by sustained growth factor treatment. Neonatal cardiomyocytes were stained with green membrane dye prior to injection and then for cleaved caspase-3 14 days after injection. Data are mean ± SEM from n ≧ 4 animals per group (** p <0.01 vs peptide alone or untethered IGF) (Example 7, below). FIG. 47 shows cell tracking using GFP and HA adenovirus. Immediately after isolation, cells were incubated in suspension with 100 MOI of GFP or hemagglutinin adenovirus for 2 hours. Cells were plated for 24 hours and flow cytometry was performed (Example 7, below). FIG. 48 is a chart showing echocardiographic data grouped 3 days after sham surgery or coronary artery ligation surgery. The measurement of fractional shortening was made 3 days after ischemia from M-mode, short axis movies. In rats that received this procedure, there was a significant reduction in cardiac function as measured by internal diameter shortening (* p <0.05; t-test). These data demonstrate our ability to perform this surgery consistently in rats and induce cardiac dysfunction (Example 7, below). FIG. 49 is a photograph showing isolation of cardiac stem cells. Cells were isolated as described, fixed in 4% paraformaldehyde, and stained with an antibody against the indicated marker, followed by a fluorescent secondary antibody (Example 7, below). FIG. 50 shows the H-NMR analysis of PK-3. 1 H-NMR spectra were acquired using a Varian Mercury VX 400 MHz NMR spectrometer (Palo Alto, Calif.) Using CDCl ₃ as the solvent. The molar ratio of 1,5-pentanediol to 1,4-cyclohexanedimethanol was obtained by obtaining the ratio of the area under peak a and peak b, respectively (Example 8, below). FIG. 51 is a schematic diagram showing the synthesis of a polyketal copolymer from 1,4-cyclohexanedimethanol (second diol) and 2,2-dimethoxypropane. The hydrolysis kinetics of the polyketal copolymer can be controlled by copolymerization (Example 8, below). FIG. 52 is a graph showing that the hydrolysis kinetics of polyketals can be adjusted by copolymerization. (A) Hydrolysis profile of polyketal copolymers PK1-PK6 in pH 4.5 buffer, and (B) Hydrolysis profile of PK1-PK6 in pH 7.4 buffer. Data are shown as mean ± standard deviation. All experiments were performed in triplicate at 37 ° C. (Example 8, below). FIG. 53 is a photograph showing an SEM image of particles formulated with PK3. SEM image of empty particles formulated by the double emulsion procedure (Example 8, below). FIG. 54 is a schematic of nitrilotriacetic acid (NTA) binding to histidine-tagged protein when loaded with nickel (Ni) (Example 9, below). FIG. 55 is a schematic illustration of a polyketal containing NTA linkers that form microspheres, where the active agent is encapsulated by the microsphere and the His-tagged protein binds to the surface of the microsphere. . B. DOGS-NTA diagram (Example 9, below). FIG. 56 is a photograph showing an SEM micrograph of 10% NTA PCADK microparticles (Example 9, below). FIG. 57 is a chart showing spectrophotometric measurements for Ni depletion in a loading solution. Various concentrations NTA- ligand (0%, 1%, 5%, 10%) a PCADK particles containing, were loaded into NiCl ₂ solution. After incubating the particles overnight with agitation, the solution was centrifuged and the supernatant was analyzed spectrophotometrically for Ni content. The supernatant of particles without NTA-ligand had a nickel concentration of about 550 mM, whereas the 1%, 5% and 10% NTA particles had about 500 mM Ni in their supernatant. This data suggests that the surface of the particles is saturated with 1% NTA-ligand inclusions. Further quantitative tests using atomic absorption spectroscopy are currently underway (Example 9, below). FIG. 58 is a photograph showing that His-tagged green fluorescent protein (GFP) is Ni-dependent. 10% NTA particles were filled with Ni, washed thoroughly, and then incubated in a 100 nM solution of His-tagged GFP. The particles were washed thoroughly with PBS and imaged using fluorescence microscopy. The (a) PCADK-NTA particles, was loaded with PBS instead of NiCl _2. Very faint fluorescence derived from GFP is seen. (B) PCADK-NTA particles loaded with NiCl ₂ show significant association with GFP (Example 9, below). FIG. 59 is a graph of the specific binding curve for 1% NTA-PCADK particles (ELISA on particles). 1% NTA-PCADK particles were filled with Ni and loaded with various concentrations of His-tagged GFP. After thorough washing after GFP loading, horseradish peroxidase (HRP) conjugated GFP antibody (α-GFP Ab) contains particles (1: 5000 dilution of α-GFP Ab and 1% goat serum). Incubated for 2 hours at room temperature with particles suspended in 1 mg particles / ml PBS-T). The particles were then washed with 3 volumes of PBS-T. Colorimetric measurements of HRP activity were performed on a plate reader using various amounts of particles per well. Absorbance was measured at 370 nm using 1-step Slow TMB-ELISA substrate (3,3 ', 5,5'-tetramethylbenzidene, Pierce) as substrate. The readings from 3, 6 and 9 minutes are compared and shown above. The specific binding curve suggests that 1% NTA particles are saturated with GFP when loaded with 60 ng GFP / mg particles (Example 9, below). FIG. 60 is a graph of specific binding curves for 10% NTA-PCADK particles (ELISA on particles). 10% NTA-PCADK particles were filled with Ni and loaded with various concentrations of His-tagged GFP. After thorough washing after GFP loading, horseradish peroxidase (HRP) conjugated GFP antibody (α-GFP Ab) contains particles (1: 5000 dilution of α-GFP Ab and 1% goat serum). Incubated for 2 hours at room temperature with particles suspended in 1 mg particles / ml PBS-T). The particles were then washed with 3 volumes of PBS-T. Colorimetric measurements of HRP activity were performed on a plate reader using various amounts of particles per well. Absorbance was measured at 370 nm using 1-step Slow TMB-ELISA substrate (3,3 ', 5,5'-tetramethylbenzidene, Pierce) as substrate. The readings from 3, 6 and 9 minutes are compared and shown above. Specific binding curves suggest that 10% NTA particles are saturated with GFP when loaded with 120 ng GFP / mg particles (Example 9, below). FIG. 61 is a graph showing that GFP binding is reversible. The GFP loaded particles were subjected to the addition of imidazole (200 mM final concentration) (competitive binding ligand to NTA-Ni complex) and the supernatant fluorescence intensity was measured as a function of time. The data suggests that GFP is most dissociated in 30 minutes in the presence of 200 mM imidazole (Example 9, below). FIG. 62 is a chart showing that GFP binding increases with the amount of NTA-Ni (Example 9, below).

(Detailed description of the invention)
(Definition)
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise stated. As used in this application, the following words or phrases have the meanings described.

  As used herein, the term “micelle” refers to a colloidal aggregate of polymer molecules having at least two different moieties associated with different properties in a liquid medium. These differences in properties can occur due to different hydrophobicity / hydrophilicity, polarity, charge or charge distribution, or other parameters that affect the solubility of the molecule. The micelles of the present invention are distinguished from and excluded from liposomes composed of bilayers.

  As used herein, the term “polymer” refers to a covalently linked arrangement of monomer molecules. This arrangement can be realized in linear or branched form. Even if the polymer is a homopolymer composed only of one type of monomer molecule, a copolymer having two or more different types of monomers bonded within the same polymer chain. It may be a coalescence. If the two different monomers are arranged in an alternating fashion, the polymer is called an alternating copolymer. In the random copolymer, the two different monomers can be arranged in any order. In the block copolymer, each type of monomer is grouped together. A block copolymer can be considered as two or more homopolymers joined together at their ends. When a polymer chain made from one monomer is grafted to the polymer chain of another monomer, a graft copolymer is formed.

  As used herein, the term “particle” or “three-dimensional particle” refers to, for example, nanoparticles and / or microparticles. According to a standard definition, the term “nanoparticle” includes only particles having at least one dimension smaller than 100 nm. Larger particles that do not meet this requirement are called “microparticles”. The present invention provides three-dimensional particles that are sized at the nanometer (nm) and micron (μm) scale. The particles of the present invention can range in size from about 50 nm to 1000 μm or from about 200 nm to 600 μm.

  As used herein, the terms “ketal” and “diol” encompass ketals and diols containing alkyl, cycloalkyl, and aryl groups. As used herein, “alkyl group” or “aliphatic group” is a linear or branched alkyl group. In one embodiment, a straight chain alkyl group is preferred. Also included in the definition of alkyl are heteroalkyl groups where the heteroatom can be nitrogen, oxygen, phosphorus, sulfur and silicon.

  As used herein, the term “cycloalkyl group” or “alicyclic group” refers to an alkyl ring structure containing at least three carbon atoms in the ring. In one embodiment, cycloalkyl groups having 5 or 6 carbons are preferred. Cycloalkyl groups also include heterocyclic rings where the heteroatoms can be nitrogen, oxygen, phosphorus, sulfur and silicon.

  As used herein, an “aryl group” or “aromatic group” refers to an aromatic aryl ring (eg, phenyl), a heterocyclic aromatic ring (eg, pyridine, furan, thiophene, pyrrole, indole and Purine), and heterocyclic rings containing nitrogen, oxygen, sulfur or phosphorus. Included in the definition of alkyl, cycloalkyl and aryl groups are substituted alkyl, cycloalkyl and aryl groups. These groups can carry one or more substitutions. Suitable substituents include, but are not limited to, halogens, amines, hydroxyl groups, carboxylic acids, nitro groups, carbonyl groups and other alkyl groups, cycloalkyl groups, and aryl groups.

  In order that the invention described herein may be more fully understood, the following description is presented.

(Composition of the present invention)
Polyketal polymer of the present invention The present invention provides a biodegradable hydrophobic polyketal polymer wherein each ketal group contains a number of ketal groups having two oxygen atoms in the polymer backbone. In one embodiment, the biodegradable hydrophobic polyketal polymer of the present invention is in the form of a solid molecule.

  Examples of suitable ketal groups include, but are not limited to, a 2,2-dioxypropyl group, a 2,2-dioxybutyl group, a 1,1-dioxycyclohexyl group, or a dioxyacetophenyl group. Also within the scope of the present invention are ketal polymers comprising aliphatic ketals, alicyclic ketals or aromatic ketals containing one or more heteroatoms (eg nitrogen, sulfur, oxygen and halides).

  In one embodiment of the invention, the polymer further comprises a compound comprising an alkyl group, an aryl group, and a cycloalkyl group. In this embodiment, the compound can be bound directly to the ketal group.

  Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl and butyl groups. Examples of suitable aryl groups include, but are not limited to, substituted or unsubstituted benzyl, phenyl or naphthyl groups (eg, 1,4-dimethylbenzene). Examples of suitable cycloalkyl groups include, but are not limited to, substituted or unsubstituted cyclohexyl, cyclopropyl, and cyclopentyl groups (eg, 1,4-dimethylcyclohexyl group).

  In a preferred embodiment, the polymer can be poly (l, 4-phenylene-acetone dimethylene ketal). This polymer can be synthesized with 2,2-dimethoxypropane and 1,4-benzenedimethanol. The polymer can also be poly (l, 4-cyclohexane-acetone dimethylene ketal), which can be synthesized with 2,2-dimethoxypropane and 1,4-cyclohexanedimethanol.

  In a further embodiment of the present invention, the polyketal polymer can be any one or more of PK1, PK2, PK3, PK4, PK5 and PK6 copolymers. These six polyketal copolymers show different hydrolysis kinetics at different pH levels. For example, at pH 4.5, PK4 hydrolyzes the fastest of these six copolymers and PK3 has the second fastest hydrolysis rate. Similarly, PK3 has faster hydrolysis kinetics than PK2 or PK5, while PK2 and PK5 have faster hydrolysis kinetics than PK1 or PK6. However, at pH 7.4, PK3 is hydrolyzed faster than PK4. By altering the copolymer percentage of the polyketal of the present invention, the hydrolysis kinetics for controllable release of the active substance can be finely tuned.

  In one embodiment, PK1 has the structure shown in Table 1 (Example 8) and is composed of at least two different monomers (ie, repeating units such as random repeating units). In one example of PK1, one type of monomer is 1,4, -cyclohexanedimethoxy having an x value of about 98% (ie, the percent component of the first monomer incorporated into the polymer). The second type of monomer is 1,5-pentanedioxy having a y value of about 2% (ie, the percent component of the second monomer incorporated into the polymer). The N value can be 3. In this embodiment, PK1 is synthesized from two types of starting compounds: 1,4-cyclohexanedimethanol and 1,5-pentanediol. The IUPAC name for PK1 is poly (cyclohexane-1,4-diyl acetone dimethylene ketal-co-1,5-pentane-acetone dimethylene ketal).

  In one embodiment, PK2 has the structure shown in Table 1 (Example 8) and is composed of at least two different monomers (ie, repeating units such as random repeating units). In one example of PK2, one type of monomer is 1,4, -cyclohexanedimethoxy having an x value of about 92% (ie, the percent component of the first monomer incorporated into the polymer). The second type of monomer is 1,5-pentanedioxy having a y value of about 8% (ie, the percent component of the second monomer incorporated into the polymer). The N value can be 3. In this embodiment, PK2 is synthesized from two types of starting compounds: 1,4-cyclohexanedimethanol and 1,5-pentanediol. The IUPAC name for PK2 is poly (cyclohexane-1,4-diyl acetone dimethylene ketal-co-1,5-pentane-acetone dimethylene ketal).

  In one embodiment, PK3 has the structure shown in Table 1 (Example 8) and is composed of at least two different monomers (ie, repeating units such as random repeating units). In one example of PK3, one type of monomer is 1,4, -cyclohexanedimethoxy having an x value of about 87% (ie, the percent component of the first monomer incorporated into the polymer). The second type of monomer is 1,5-pentanedioxy having a y value of about 13% (ie, the percent component of the second monomer incorporated into the polymer). The N value can be 3. In this embodiment, PK3 is synthesized from two types of starting compounds: 1,4-cyclohexanedimethanol and 1,5-pentanediol. The IUPAC name for PK3 is poly (cyclohexane-1,4-diyl acetone dimethylene ketal-co-1,5-pentane-acetone dimethylene ketal).

  In one embodiment, PK4 has the structure shown in Table 1 (Example 8) and is composed of at least two different monomers (ie, repeating units such as random repeating units). In one example of PK4, one type of monomer is 1,4, -cyclohexanedimethoxy having an x value of about 97% (ie, the percent component of the first monomer incorporated into the polymer). The second type of monomer is 1,4-butanedioxy having a y value of about 3% (ie, the percent component of the second monomer incorporated into the polymer). The N value can be 2. In this embodiment, PK4 is synthesized from two types of starting compounds: 1,4-cyclohexanedimethanol and 1,4-butanediol. The IUPAC name for PK4 is poly (cyclohexane-1,4-diyl acetone dimethylene ketal-co-1,4-butane-acetone dimethylene ketal).

  In one embodiment, PK5 has the structure shown in Table 1 (Example 8) and is composed of at least two different monomers (ie, repeating units such as random repeating units). In one example of PK5, one type of monomer is 1,4, -cyclohexanedimethoxy having an x value of about 85% (ie, the percent component of the first monomer incorporated into the polymer). The second type of monomer is 1,6-hexanedioxy having a y value of about 15% (ie, the percent component of the second monomer incorporated into the polymer). The N value can be 4. In this embodiment, PK5 is synthesized from two types of starting compounds: 1,4-cyclohexanedimethanol and 1,6-hexanediol. The IUPAC name for PK5 is poly (cyclohexane-1,4-diyl acetone dimethylene ketal-co-1,6-hexane-acetone dimethylene ketal).

  In one embodiment, PK6 has the structure shown in Table 1 (Example 8) and is composed of at least two different monomers (ie, repeating units such as random repeating units). In one example of PK6, one type of monomer is 1,4, -cyclohexanedimethoxy having an x value of about 87% (ie, the percent component of the first monomer incorporated into the polymer). The second type of monomer is 1,8-octanedioxy having a y value of about 13% (ie, the percent component of the second monomer incorporated into the polymer). The N value can be 6. In this embodiment, PK6 is synthesized from two types of starting compounds: 1,4-cyclohexanedimethanol and 1,8-octanediol. The IUPAC name for PK6 is poly (cyclohexane-1,4-diyl acetone dimethylene ketal-co-1,8-octane-acetone dimethylene ketal).

  Said PK1-PK6 copolymer may be synthesized using an acetal exchange reaction by copolymerizing 1,4-cyclohexanedimethanol with either butanediol, pentanediol, hexanediol or octanediol. As shown in Table 1 of Example 8 below, PK1 was synthesized using monomer A (1,4-cyclohexanedimethanol) and monomer B (1,5-pentanediol); PK2 Was synthesized using monomer A (1,4-cyclohexanedimethanol) and monomer B (1,5-pentanediol); PK3 was monomer A (1,4-cyclohexanedimethanol) And monomer B (1,5-pentanediol); PK4 uses monomer A (1,4-cyclohexanedimethanol) and monomer B (1,4-butanediol) PK5 was synthesized using monomer A (1,4-cyclohexanedimethanol) and monomer B (1,6-hexanediol); PK6 was synthesized using monomer A (1,4 -Cyclohexane dimethano Le) and the monomer B (1,8-synthesized using octanediol).

  In another embodiment of the present invention, the PK1, PK2, PK3, PK4, PK5 and PK6 copolymers of the present invention can be combined to alter or fine tune the release rate profile of the attached active agent. For example, polyketals with fast hydrolysis kinetics (eg, PK4 or PK3) can be mixed with polyketals with slower hydrolysis kinetics (eg, PK1 or PK6) and co-administered (co- administrator). The active agent added to PK4 or PK3 is released rapidly in the subject, providing the subject with an immediate release (IR) dose of the active agent, while the active agent added to PK1 or PK6 is more It is released at a slow rate, allowing a gradual or extended release (ER) of the active substance in the subject.

  In one embodiment of the present invention, the biodegradable hydrophobic polyketal polymer is (1) a plurality of ketal groups, each ketal group having two oxygen atoms in the polymer skeleton. A ketal group, and (2) a linker. This ketal group may comprise a 2,2-dioxypropyl group.

Biodegradable polyketal particles of the present invention The present invention further provides biodegradable particles comprising the polyketal polymer of the present invention. The size of the particles can vary. For example, biodegradable particles can be made on the nanometer (nm) or micron (μm) scale and can form, for example, nanoparticles or microparticles. The particles of the present invention can range in size from about 50 nm to 1000 μm. In one embodiment, the particles range in size from about 200 nm to 600 μm. In another embodiment, the particles range in size from about 1 μm to 10 μm, 10 μm to 20 μm, 20 μm to 30 μm, 30 μm to 40 μm, or 40 μm to 50 μm. In yet another embodiment, the particles range in size from about 1 μm to 50 μm. A preferred particle size is between about 50 nm and 1000 nm, more preferably between about 200 nm and 600 nm. Another preferred particle size is about 30 μm.

  The preferred size of a suitable polyketal polymer for forming biodegradable particles is between about 0.5 kDa and about 2 MDa, more preferably between 1 kDa and about 150 kDa, most preferred Is between about 4 kDa and about 6 kDa. In accordance with the present invention, the number of monomers in the polymer can range from about 2 to about 20,000, preferably from about 10 to about 1,000, more preferably from about 10 to about 50.

  The polyketal polymer of the present invention is hydrolyzed in an aqueous solution into low molecular weight, water soluble alcohols and ketones. For example, the degradation of poly (alkyl-acetone dimethylene ketal) is acid sensitive and has a half-life of 102.0 hours at pH 7.4 and 35.0 hours at pH 5.0. The advantage of a ketal bond within the backbone is that this ketal bond degrades at pH 5.0 within 1-2 days under the phagosomal acidic conditions. Thus polyketals can also be used to target the acidic environment of tumors, inflammation and phagolysosomes. This degradation does not produce acidic degradation products. Therefore, the ketal polymer is suitable for biological use.

  In accordance with the practice of the present invention, the polyketal polymer particles may further comprise one or more active substances.

  In one embodiment of the present invention, the biodegradable particle of the present invention is (a) a biodegradable hydrophobic polyketal polymer containing a plurality of ketal groups, and each ketal group is contained in the polymer skeleton. A biodegradable hydrophobic polyketal polymer having two oxygen atoms, and (b) a linker. Suitable polymers for forming the particles of the present invention include PCADK and PK1-PK6.

  In one embodiment of the invention, the polyketal polymer particles described above may further comprise one or more linkers that can be bound to the active agent. Multiple linkers of the same or different types can be attached to the polymer particles. These linkers are attached to the surface of the particle. That is, these linkers are exposed to a solvent or aqueous solution around the particles.

  In one embodiment, the biodegradable particles of the invention comprise (a) one or more PK1, PK2, PK3, PK4, PK5 or PK6 (which may optionally include a linker), and (b) Contains active substance. In another embodiment of the invention, the biodegradable particles of the invention comprise (a) PCADK (which may optionally include a linker), and (b) an active agent.

  In accordance with the practice of the invention, the particles may comprise a single population or a mixed population of polyketals.

Active Substances of the Invention As used herein, the term “active substance” refers to proteins, peptides, nucleic acids (DNA or RNA) or organic molecules, and other synthetic nucleic acid molecules, siRNA molecules, or antisense molecules. Point to. The active substance can be a therapeutic agent, a prophylactic agent or a diagnostic agent. This therapeutic agent is an immunomodulator (eg, RIG-1 or TLR, or a specific ligand for a C-type lectin (eg, dectin-1 and DC-SIGN) or caterpillar protein, or a combination of specific TLR ligands, or DC. And a synthetic molecule or siRNA that inhibits the regulatory signaling network by macrophages). The active agent is further a protein (eg, recombinant protein), peptide, carbohydrate, nucleic acid, small molecule (eg, kinase inhibitor, phosphatase inhibitor, cytokine inhibitor, small molecule antioxidant mimic, receptor blocker, cytoskeleton reconstitution) Inhibitors, small molecule receptor activators), imaging agents, vaccine antigens, DNA vaccines, or vaccines themselves (eg, influenza vaccines). Ultimately, the active agent targets or stimulates a subset of DCs (including Langerhans cells, skin DCs, bone marrow DCs, intestinal DCs, plasmacytoid DCs), or a subset of monocytes and macrophages Or antibodies that modulate or inhibit. Thus, the surface of these particles is targeted to a target group (eg, an antibody against a subset of dendritic cells, or a protein that stimulates a subset of dendritic cells or macrophages (eg, CD40L, DEC-205, CD11c, Langerin, MARCO, 33D1). Etc.)) and the like.

  Examples of suitable active agents include, but are not limited to: (1) TLRs (eg, TLR-2, TLR-3, TLR-4, TLR-5, TLR-7, TLR-8 , TLR-9, TLR-10, and TLR-11) agonists and antagonists, (2) Schistosomiasis egg antigen (SEA) activated agonists and antagonists, (3) these types of receptors A molecule that stimulates or inhibits the expression or activity of a component of an intracellular signaling pathway that transmits a signal resulting from the activation of any of the above, (4) a transcription factor that is induced or stabilized by one or more of these signaling pathways And (5) regulatory pathways in dendritic cells, macrophages or antigen presenting cells Bitter.

  Examples of agonists include peptidoglycan (O. Takeuchi et al., 1999 Immunity 11: 443-451) or zymosan (Dillon et al., 2006 J. Clin. Invest. 116 (4): 916-28 A. Ozinsky et al., 2000 Proc. Natl. Acad. Sci. USA 97: 13766-13771), but is not limited thereto. Agonists also include bacterial lipopeptides (eg, diacylated and triacylated lipopeptides), lipoteichoic acid, lipoarabinomannan, phenol-soluble modulin, glycoinositol phospholipids, glycolipids, porin, Leptospira Also included are atypical LPS from interrogns or Porphyromonas gingivalis, or HSP70 (for review, see K Takeda et al., 2003 Annu. Rev. Immunol. 21: 335-376). The agonist can be an isolated and / or highly purified molecule. Such agonists include whole molecules or fragments thereof, or naturally occurring or synthetic agonists. Examples include the non-toxic form of cholera toxin (Braun et al., J. Exp. Med. 189: 541-552, 1999), the specific form of Candida albicans (d'Ostiani et al., J. Exp. Med. 1911661 -1674, 2000), or P.I. gingivalis LPS (Pulendran et al., J. Immunol 167: 5067-5076, 2001).

  Examples of bacterial lipopeptides include bacterial cell wall lipopeptides (eg, diacylated and triacylated lipopeptides) that differ in the N-terminal cysteine fatty acid chain. For example, diacylated lipopeptides include macrophage activated lipopeptides 2 kilodalton from Mycoplasma fermentans or fragments thereof or synthetic analogs (eg, MALP2, Pam2CSK4, Pam2CGNNDESNISFEKK, and Pam2CGNNDESNISFEKK-SK4). As the triacylated lipopeptide, Pam3cys {S- [2,3-bis (palmitoyloxy)-(2-RS) -propyl] -N-palmitoyl-R-Cys-S-Ser-Lys4-OH)} (Takeuchi et al., 2001 International Immunology 13: 933-940).

  In certain embodiments, the agonist specifically acts on a receptor that is bound by TLR-2 or SEA (for SEA, see MacDonald et al., J. Immunol. 167: 1982-1988, 2001). Again, the agonist can be a natural ligand, a biologically active fragment thereof, or a small or synthetic molecule, but is not limited thereto. Other useful agonists include non-toxic forms of cholera toxin (Braun et al., J. Exp. Med. 189: 541-552, 1999), specific forms of Candida albicans (d'Ostani et al., J. Exp. Med). 191: 1661-1674, 2000), or Porphyromonas gingivalis LPS (Pulendran et al., J. Immunol. 167: 5067-5076.2001). These substances cannot induce IL-12 (p70) and cannot stimulate a Th2-like response.

  The agonist can be an agonist of TLR-4 (which biases the immune response to a Th response (eg, TH1)), taxol, a rous sarcoma virus-derived fusion protein, an MMTV-derived envelope protein, derived from Chlamydia pneumoniae Hsp60 or host-derived Hsp60 or Hsp70. Other host factors that act on TLR-3 include type III repeat extra domain of fibronectin, oligosaccharides of hyaluronic acid, polysaccharide fragments of heparin sulfate, and fibrinogen. Many synthetic compounds, such as imidazoquinolines (imiquimod and R-848), loxoribine, bropirimine, and other compounds structurally related to nucleic acids, act as agonists of TLR-7.

  Further examples of suitable active substances include ERK, c-Fos, Foxp3, PI3 kinase, Akt, JNK, p38, NF-Kb, STAT 1, STAT2, IRF3, IRF7, IFN-α signaling inhibitors or combinations thereof Is mentioned. Suitable active substances may further include inhibitors of SOCS 1-7 protein. Such inhibitors can be small molecules, or peptides, proteins or nucleic acids (eg, siRNA or antisense).

  The agonist may be an endogenous ligand or an exogenous ligand, many of which are known in the art. The following novel screening methods (especially methods that feature detection of TLR binding or activation) can be performed using other ligands (naturally occurring molecules, fragments or derivatives thereof, antibodies, other peptides or proteins). Which can be either a complex containing or a synthetic ligand). For example, exogenous ligands for TLR-2 include LPS (lipopolysaccharide; a component of the outer membrane of Gram-negative bacteria), zymosan, a yeast particle, bacterial peptidoglycan, lipoproteins derived from bacteria and mycobacteria, and GPI anchors from Trypanosoma cruzi are included; endogenous ligands include heat shock (or “stress”) proteins (eg, Hsp60 from, for example, bacterial or mycoplasma pathogens) and surfactant protein A. Exogenous ligands for TLR-3 include poly (I: C) (virus dsNRA); exogenous ligands for TLR-4 include LPS and RS virus (endogenous ligands include stress Proteins (eg, Hsp60 or Hsp70), saturated fatty acids, unsaturated fatty acids, hyaluronic acid and fragments thereof, and surfactant protein A). Flagellin is an exogenous ligand of TLR-5. CpG (cytosine-guanine repeat) DNA and dsDNA are the exogenous and endogenous ligands of TLR-9, respectively. Zuany-Amorim et al., Nature Reviews 1: 797-807, 2002, and Takeda et al., Ann. Rev. Immunol. 21: 355-376, 2003.

  Further examples of suitable active substances include (a) substances that inhibit the expression or activity of AP-1 transcription factor in dendritic cells, (b) substances that inhibit the expression or activity of AP-1 transcription factor Dendritic cells treated in culture, or syngeneic T cells stimulated in a culture containing dendritic cells treated as described in (c) (b). The transcription factor may include c-fos, fos-B, Foxp3, or c-jun (the transcription factor or any component of the pathway described herein (these components Substances that inhibit expression) are c-fos, fos-B, Foxp3, or c-jun expression (or kinase, phosphatase, or expression of other components of the signaling pathway). ) Can be antisense oligonucleotides or RNAi molecules. The inhibitory active substance discussed in connection with the biodegradable particles of the invention can also be an antibody (or a variant thereof (eg, a single chain antibody or a humanized antibody); preferably the antibody is monoclonal Antibody).

  In another embodiment, the active agent is an antagonist (eg, inhibitor or suppressor) of an intracellular pathway that attenuates TLR2 signaling or activation. Examples of the antagonist include Gram negative LPS, taxol, RSV fusion protein, MMTV envelope protein, HSP60, HSP70, fibronectin type III repeat extra domain A, hyaluronic acid oligosaccharide, heparin sulfate oligosaccharide fragment, fibrinogen and flagellin (For example, see K Takeda et al., 2003 Annu. Rev. Immunol. 21: 335-376 for a review).

  In a further embodiment, the active agent is an antagonist of an intracellular pathway that attenuates SEA signaling or activation. In one other embodiment, the molecule is an antagonist of the JNK 1/2 pathway. In another embodiment, the molecule is CpG DNA that activates p38 and ERK (AK Yi et al., 2002 The Journal of Immunology 168: 4711-4720).

  In another embodiment, the active agent is an inhibitor of ERK 1/2 that can inhibit dendritic cell maturation and thus increase IL12 and Th1 responses. Examples of such molecules include, but are not limited to, PD98059 and U0126 (A. Puig-Kroger et al., 2001 Blood 98: 2175-2182).

  In another embodiment, the active agent inhibits c-fos signaling and thus increases IL12 and Th1 responses. Such molecules include c-fos DEF domain mutants or any polypeptide having a DEF domain mutation (L.O. Murphy et al., 2002 Nature Cell Biology 4: 556-564 and Appendix Information 1- 3)), including rat Fra-1 and Fra-2; mouse FosB, JunD, c-Jun, c-Myc, and Egr-1; and human JunB, N-Myc, and mPerl. Can be mentioned.

  The active agent can include nucleic acids such as DNA, RNA, antisense oligonucleotides or siRNA. Suitable examples of DNA include therapeutic genes. An example of a therapeutic gene is a suicide gene. These are gene sequences whose expression produces proteins or agents that inhibit tumor cell growth or tumor cell death. Suicidal genes include genes encoding enzymes, oncogenes, tumor suppressor genes, genes encoding toxins, genes encoding cytokines, or genes encoding oncostatin. The purpose of the therapeutic gene is to inhibit the growth of cancer cells, or to kill cancer cells, or to directly or indirectly inhibit the growth of cancer cells or to kill cancer cells or other It is to produce cytotoxic substances.

  Suitable oncogenes and tumor suppressor genes include neu, EGF, ras (including H, K, and N ras), p53, retinoblastoma tumor suppressor gene (Rb), Wilms tumor gene product, phosphotyrosine phosphatase ( PTPase), and nm23. Suitable toxins include Pseudomonas exotoxin A and exotoxin S; diphtheria toxin (DT); E. coli LT toxin, Shiga toxin, Shiga-like toxin (SLT-1, -2), ricin, abrin, supporin, and gelonin.

  The active substance can include an enzyme. Suitable enzymes include thymidine kinase (TK), E.I. xanthine-guanine phosphoribosyltransferase (GPT) gene from E. coli or E. coli. E. coli cytosine deaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT).

  The active substance can include a cytokine. Suitable cytokines include interferon, GM-CSF, interleukin, tumor necrosis factor (TNF) (Wong G et al., Science 1985; 228: 810); WO93223034 (1993); Horisberger M. et al. A. Et al., Journal of Virology, May 1990, 64 (3): 1171-81; Li YP et al., Journal of Immunology, February 1, 1992, 148 (3): 788-94; T.A. Et al., Transplantation, August 1993, 56 (2): 399-404); (Brevario F. et al., Journal of Biological Chemistry, November 5, 1992, 267 (31): 22190-7; Espinosa-Delgado I. , Et al., Journal of Immunology, November 1, 1992, 149 (9): 2961-8; Algate PA, et al., Blood, May 1, 1994, 83 (9): 2459-68; H. et al., Anals of Hematology, June 1994, 68 (6): 293-8; Martinez OM et al., Transplantation, May 1993, 55 (5): 1159-66.

  The active substance can comprise a growth factor. Growth factors include transforming growth factor α (TGF-α) and β (TGF-β), cytokine colony stimulating factor (Shimane M. et al., Biochemical and Biophysical Research Communications, Feb. 28, 1994, 199 ( 1): 26-32; Kay AB et al., Journal of Experimental Medicine, Mar. 1, 1991, 173 (3): 775-8; de Wit H et al., 1994 Feb., 86 (2): 259- 64; Sprecher E. et al., Archives of Virology, 1992, 126 (l-4): 253-69).

  Furthermore, examples of the active substance include catalase, superoxide dismutase, glutathione peroxidase, and nitric oxide synthase, which are proteins.

  The active substances of the present invention may be naturally occurring, synthetic or recombinantly produced, including any microbial or viral component or derivative thereof (microbial cell or viral Any component that is part of or produced by the structure (cell wall, coat protein, extracellular protein, intracellular protein, any toxic or non-toxic compound, carbohydrate, protein-carbohydrate complex, or microorganism Including, but not limited to, any other component of a cell or virus)). The microbial cell or virus can be pathogenic.

Linkers of the Invention The polyketals of the invention (eg, in particle embodiments) may further comprise one or more linkers. The linker may be of the same type or may comprise a mixture of different types attached to the polyketal polymer. This linker can be attached to the surface of the polyketal polymer. For example, the linker can be exposed to a solvent or aqueous solution around the particle.

  The linker may range in size from 1-1000 nm or 100-200,000 Da. In one embodiment of the invention, the linker is 1-10 nm, 10-50 nm, 50-100 nm, 100-200 nm, 200-300 nm, 300-400 nm, 400-500 nm, 500-600 nm, 600-700 nm, 700- It can range in size from 800 nm, 800-900 nm and 900-1000 nm. In another embodiment of the invention, the linker is 100-10,000 Da, 10,000-50,000 Da, 50,000-100,000 Da, 100,000-150,000 Da and 150,000-200,000 Da. Range of sizes. In yet another embodiment of the invention, the linker may be about 200-300 Da, 10,000 Da, 40,000 Da, 64,000 Da, or 150,000 Da in size.

  The linker can be a protein, polypeptide, nucleic acid or compound. Examples of protein or polypeptide linkers include, but are not limited to, amino acid sequences such as arginine-aspartate-glycine (RDG), hemoglobin, glutathione-S-transferase (GST), streptavidin or antibodies. Examples of nucleic acid linkers include, but are not limited to, RNA, DNA (eg, ssDNA such as 4-50 nucleic acid long tracts of poly A or poly T sequences) or synthetic analogs. Examples of compounds that can be used as linkers include, but are not limited to, polycarboxylic acids (eg, polyacrylic acid) or nitrilotriacetic acid (NTA) ligands, bipyradol and EDTA. . The chelator binds to a metal ion (eg, nickel, zinc, copper, etc.), which in turn promotes binding of the active agent to the linker. This linker anchors or combines the active agent (eg, via a non-covalent bond) to the polyketals of the present invention.

  The linker may be attached to an active agent, including but not limited to proteins, peptides, nucleic acids and / or carbohydrates. In one embodiment, the protein, peptide and / or carbohydrate further comprises a histidine tag. The histidine tag can contain about 2-8 histidines. For example, in one embodiment, the histidine tag comprises about 6 histidines. The protein so bound to the linker can be a therapeutic protein, a prophylactic protein or a diagnostic protein.

  In accordance with the practice of the invention, the therapeutic protein can be an immunomodulatory protein. For example, the immunomodulatory protein may include, but is not limited to: (a) a ligand for any of TLR2, 3, 4, 5, 7, 8, 9, 10 and 11 or Combinations thereof, (b) inhibitors of regulatory pathways in dendritic cells, macrophages or antigen presenting cells, and (c) RIG-1, any C-type lectin (including dectin-1 and DC-SIGN) or caterpillar protein Ligand for. Inhibitors of the regulatory pathway include: (a) ERK, c-Fos, Foxp3, PI3 kinase, Akt, JNK, p38, NF-Kb, STAT1, STAT2, IRF3, IRF7, IFN-α signaling; or (b) SOCS1 2, 3, or other SOCS protein inhibitors may be mentioned.

  Further suitable examples of proteins that bind to the linker include growth factors, cytokines, antioxidant enzymes, antibodies, erythropoietin (EPO), receptor ligands.

  In one embodiment, the polyketals of the present invention (eg, either PCADK or PK1-PK6) comprise an NTA linker. This NTA linker can be loaded with nickel ions to produce an NTA-Ni linker that binds to histidine-tagged proteins.

(Micelle of the present invention)
The present invention further provides biodegradable cross-linked micelles comprising multiple polymers of the present invention. The polymer can be crosslinked with an external crosslinking agent. The external cross-linking agent used in the present specification is a drug that is not introduced into the polymer chain. The advantage of using an external agent is that the cross-linking reaction is faster compared to reactions in which only cross-linkable moieties within the polymer are used. The external cross-linking agent also reduces the chance that the encapsulated protein is destroyed.

  In one aspect of the invention, suitable crosslinkers are compounds that contain at least two thiol groups. Examples of suitable crosslinkers include, but are not limited to, ethylene glycol dithiols, aliphatic dithiols, dithiols linked by ketal linkages, and diamine-containing molecules. The advantage of the thiol group is that it is sensitive to reducing conditions and thus allows easy degradation of the micelles. Other crosslinking strategies include amines, esters, carbonates, thioesters, Schiff bases, vicinal diols, alkenes and alkynes, ketals, ketal orthoesters, thio-ketals, thio-orthoesters, silly-ketals, phenyl boronic acid-diols Examples include, but are not limited to, complexes, carbon-carbon bonds, sulfones, functional groups including phosphates, azides, enzymatically cleavable bonds, and crosslinking with urethane. Schiff bases, thiols and ketones are preferred in one embodiment of the present application (O'Reilly et al., 2005 Chem. Mater., 17 (24): 5976-5988; Hanker et al., 2005 Science 309 (5738): 1200-05. Le, Z. et al., 2005 Langmuir 21 (25): 11999-12006; Example 1, Figure 9).

  In another aspect of the present invention, the external cross-linking agent includes an antigen. Examples of suitable antigens include but are not limited to proteins or peptides. The antigen can be naturally occurring, chemically synthesized or can be produced recombinantly. Specific examples of suitable protein or peptide antigens include HIV antigen (eg, gpl20 protein (or fragment thereof)), TAT protein (or fragment thereof), NEF protein (or fragment thereof), HCV protein (or fragment thereof) ), And env proteins (or fragments thereof). Preferred antigens include gpl20 peptide antigens that have been chemically synthesized and modified to include four additional cysteine residues to create additional disulfide bonds therein. Any antigen having a crosslinkable group can be used. Antigens with little or no crosslinkable groups can be modified to include crosslinkable thiol groups, azides, alkynes, amines, maleimides, vinyl sulfones, ketones, hydrazines and thioesters (eg chemically Can be modified).

  The polymer used for micelle formation can be a homopolymer or a copolymer (eg, a block copolymer or a graft polymer). Examples of suitable polymers include PEG block copolymers (eg, PEG-polyamino acids (eg, PEG-polylysine, PEG-polyglutamic acid, PEG) synthesized by atom transfer polymerization in which PEG acts as an initiator. -Polyaspartic acid or PEG-polyarginine); PEG-polyester, PEG-polyurethane, PEG-PPO, modified or unmodified, PEG-polyacrylate or PEG-polymethacrylate) It is not limited to. In order to promote cross-linking of the polymer, the polymer may contain chemical groups (amines, esters, carbonates, thioesters, Schiff bases, vicinal diols, alkenes and alkynes, ketals, ketal orthoesters, thio-ketals, thio-orthos. Esters, silly-ketals, phenyl boronic acid-diol complexes, carbon-carbon bonds, sulfones, phosphate-containing functional groups, azides, enzymatically cleavable bonds, and urethanes, but are not limited to, Schiff bases , Thiols and ketones are preferred in one embodiment of the invention). These groups can be introduced by chemical reactions known in the art (eg, among others, Michael addition or acylation) (Example 1, FIG. 9). In one preferred embodiment, the modified polymer is PEG-polylysine thiopyridal (FIG. 9).

  The polymethacrylate block or polyacrylate block may include modifications to allow assembly with vaccine components and cross-linking. For example, the polyacrylate block can be a block copolymer consisting of polydimethylamino-acrylate-poly-glycidyl acrylate. Homopolymers of random copolymers composed of various acrylate or methacrylate monomers that have the ability to form micelles using vaccine components are also within the scope of the present invention.

  In another aspect of the invention, the micelles can further comprise one or more active substances. Examples of suitable active substances are mentioned herein above.

  In accordance with the practice of the present invention, the interaction between the micelle and the active substance can be electrostatic or hydrophobic, or depends on hydrogen bond formation or molecular recognition, depending on the type of polymer and substance. Can occur. The surface of the micelles is targeted to a targeting group (eg, antibodies against dendritic cells, or proteins that stimulate a subset of dendritic cells or macrophages (eg, CD40L, DEC-205, CD11c, Langerin, MARCO, 33D1, etc.)). It can be modified to include.

  In one embodiment, the micelles are designed to deliver peptide antigens and immunomodulatory molecules to antigen presenting cells (APC). In this embodiment, the micelle comprises an immunomodulatory molecule, a peptide antigen and a copolymer. The peptide antigens enable the peptide antigens to be efficiently encapsulated into peptide cross-linked micelles (PCM) and also stabilize the PCM against degradation by serum components Acts as

  In another embodiment, the micelles are immunomodulatory agents (multiple TLR ligands, or synthetic compounds or siRNAs that modulate signaling networks within a cell (eg, a dendritic cell or other antigen presenting cell)). Can be used to encapsulate peptide antigens or protein antigens together. Since dendritic cells and macrophages firmly internalize nanometer-sized substances by phagocytosis, the micelles target these cells by having nanometer dimensions. The micelles are cross-linked by a cross-linking agent containing a disulfide bond, which stabilizes the micelles against degradation induced by serum proteins. In a further embodiment of the invention, the micelle has a size of 5-50 microns.

  After phagocytosis, the biodegradable particles and micelles of the invention are degraded and the encapsulated material (eg, peptide or antigen, and immunostimulant [eg: ISS DNA, TLR 7/8, TLR 3 ligand] (Eg, ss RNA), TLR 2 ligands, and inhibitors of regulatory pathways (eg, ERK, c-Fos or Foxp3 pathways)) are released into dendritic cells or macrophages, and the immunomodulatory agent is an antigen Presenting cells are induced to secrete various cytokines. This combination of signals results in optimal T cell activation and inhibition of regulatory T cells and dendritic cells.

  The micelles of the present invention comprise immunomodulators [eg: ISS DNA, TLR 7/8 ligand (eg ss RNA), TLR 2 ligand, and inhibitors of regulatory pathways (eg ERK, c-Fos or Foxp3, PI3 kinase, Akt, an inhibitor of SOCS 1-7 protein), or siRNA molecules or antisense molecules that inhibit such regulatory pathways] along with active agents (eg, vaccines composed of antigens from related pathogens) .

  The present invention also provides a method for reversibly modifying these proteins so that the proteins have the appropriate charge to be encapsulated in the micelles. This strategy is based on reacting the amine group of the protein with a compound to generate additional negative charges for all amine groups, making the protein negative. The modified protein is then encapsulated in the micelles, crosslinked, and then the pH is lowered to remove the inserted compound from the protein. In one embodiment of the present invention, in the compound, the amine group is cis-aconityl. This group adds a negative charge to each amine group and can be released at ph 4.0.

  An example for a targeting strategy involves synthesizing a heterobifunctional PEG having a DNA binding domain at one end and another end that can be attached to a protein. This PEG chain is then attached to a protein and then assembled into preformed micelles containing immunostimulatory DNA. Examples of DNA binding domains include acridine or polyacridine. Examples of targeting ligands include galactose, mannose phosphate, mannose, peptides, and antibodies.

Further modifications of the biodegradable particles and micelles of the present invention The biodegradable particles or micelles further comprise (1) a specific subset of DCs or macrophages or monocytes (Langerhans cells, skin DCs, bone marrow DCs, plasmacytoids) Specific receptors on (including DC), or (2) antibodies or other targeting specific receptors on antigen presenting cells (eg DEC205, Langerin, DC-SIGN, dectin-1, 33D1, MARCO) Can be modified to incorporate

Pharmaceutical Composition of the Invention The present invention provides a pharmaceutical composition comprising a polymer of the invention, which may optionally include a linker. In one embodiment, the present invention provides a pharmaceutical composition comprising a particle of the present invention, which may optionally include a linker. In another embodiment, the present invention provides a pharmaceutical composition comprising a micelle of the present invention, which may optionally include a linker. The polymers of the present invention may further comprise an active substance, whether in the form of particles or micelles. For example, in one embodiment, the present invention provides a pharmaceutical composition comprising PCADK, PK1, PK2, PK3, PK4, PK5 and / or PK6 particles having a linker attached to an active agent.

Methods of the Invention Methods for Producing the Particles and Micelles of the Invention The invention provides methods for producing the particles of the invention. In one embodiment, the method comprises: a) forming a hydrophobic polymer of a ketal and a diol or unsaturated alcohol; b) polymer particles of the polymer of a) in the presence of one or more active substances. And thereby encapsulating the material. Examples of suitable chemistries for forming ketal and diol or unsaturated alcohol hydrophobic polymers include acetal exchange reactions (Heffernan MJ and Murthy N) using single or double emulsions and acyclic diene metathesis. , 2005 Bioconjug.Chem.16 (6): 1340-2; Jin RA., 2000 Biomaterials.21 (23): 2475-90: Wagener KB and Gomez FJ, "ADMET Polymerization", Encyclopedia. of Materials: Science and Technology, edited by EJ Kramer and C. Hawker, Elsevier, Oxford, 5, 48 (200 2)).

  Examples of suitable ketals for this process include, but are not limited to, 2,2-dimethoxypropane, 2,2-dimethoxybutane, 1,1-dimethoxycyclohexane or dimethoxyacetophenol. Also within the scope of the present invention are ketal polymers comprising aliphatic ketals, alicyclic ketals or aromatic ketals containing one or more heteroatoms (eg, nitrogen, sulfur, oxygen and halides). .

  In accordance with the practice of the invention, the diol can be any of alkyl diols, aryl diols and cycloalkyl diols.

  Suitable examples of diols include 1,4-benzenedimethanol, 1,4-cyclohexanedimethanol, 1,5-pentane diol, 1,4-butane diol, or 1,8-octane diol. It is not limited to.

  In one embodiment, the present invention provides a method for producing the particles of the present invention comprising: (a) forming a PCADK polymer; and (b) the presence of one or more active agents. The process includes forming PCADK particles, thereby producing the particles of the present invention. The PCADK polymer may include a linker as required.

  In one embodiment, the present invention provides a method for producing the particles of the present invention, the method comprising: (a) forming a PK1, PK2, PK3, PK4, PK5 and / or PK6 polymer; And (b) forming one or more particles of PK1, PK2, PK3, PK4, PK5 and / or PK6 in the presence of one or more active substances, thereby forming the particles of the present invention. Include. The PK1-PK6 polymer may include a linker as necessary.

  The micelles of the present invention can be produced in a two step process. Initially, the polymer of interest can be contacted with a liquid (polar or nonpolar liquid depending on the polymer used) under conditions suitable to form micelles. After the formation of micelles, the micelles can be crosslinked with an external crosslinking agent to produce the biodegradable micelles of the present invention.

(Method for using the composition of the present invention)
The invention further provides, for example, the particles of the invention to a subject to deliver an active agent to treat a subject afflicted with a disease or disorder (eg, alleviate symptoms associated with the disease). Methods are provided for delivering the active agents of the invention through the micelles of the invention. The particles (eg, made from PCADK, PK1, PK2, PK3, PK4, PK5, or PK6 polymers) or micelles are then released into the subject so as to release the active agent for delivery to the subject. Can be degraded or dissolved. The particles or micelles of the present invention can be designed to have a variable degradation rate at various pH ranges and release the active substance according to the desired profile. For example, particles formed from PK3 polymer degrade faster than particles formed from PK6 polymer, releasing the active substance.

  In one embodiment of the invention, the particles or micelles of the invention used to deliver the active agent further comprise a linker. In further embodiments, the particles formed from the PCADK polymer further comprise a linker (eg, NTA) attached to the active agent that is used to deliver the active agent to the subject.

  The disease or disorder affecting the subject can be any of HIV, malaria, TB, SARS, anthrax, Ebola, influenza, avian influenza and HCV. The additional disease or disorder can be any of the disorders or complications associated with infectious diseases, autoimmune diseases, allergic diseases, transplantation, diabetes and cancer. Examples of autoimmune diseases include lupus, rheumatoid arthritis, psoriasis, asthma and COPD.

  The active agent is delivered via the particles of the invention by a variety of means of administration, including but not limited to intravenous, subcutaneous, intramuscular, oral and inhalation means. The most effective mode of administration and dosage regimen for the compositions of the present invention is the exact location of the disease or disorder being treated, the severity and course of this disease or disorder, the health of the subject and its response to treatment, and its Depends on the judgment of the treating physician. Therefore, the dose of this molecule should be adjusted for the individual subject.

The correlation between doses for animals of various sizes and species and humans based on mg / m ² (surface area) is described by Freireich, E .; J. et al. Et al., Cancer Chemother. Rep. 50 (4): 219-244 (1966). Adjustments in the dosing regimen can be made to optimize tumor cell growth inhibition and killing responses (e.g., doses can be divided, administered on a daily basis, or Depending on the situation, it can be reduced proportionally (eg, several divided doses can be administered daily or can be reduced proportionally depending on its specific treatment situation). It is clear that the dose of the composition of the invention required to achieve the treatment can be further reduced by schedule optimization.

  As used herein, the term “subject” includes any human, any animal (eg, horse, pig, cow, mouse, dog, cat, and avian subject), cell or cellular tissue. obtain. In accordance with the practice of the invention, the active agent can be delivered or administered to the subject (using the compositions of the invention) before, after, or during the onset of the disease or disorder.

  The present invention provides a method for modulating an immune response by administering an active agent through the particles or micelles of the present invention. In one embodiment of the invention, the particles or micelles used in the method of the invention further comprise a linker. For example, in the methods of the present invention, the immune response can be biased toward a Th immune response in a TLR-dependent manner by delivering or administering to the subject particles or micelles of the present invention containing the desired active agent. In one embodiment, the TLR-expressing cell is contacted with a substance (delivered by the particles or micelles of the invention) that results in a bias against a Th immune response (eg, a Th0, Th2, or T regulatory cell immune response) . For example, a substance that activates TLR-2, ERK 1/2, or c-fos (eg, a natural ligand, a biologically active fragment thereof, or a small or synthetic molecule).

  As described above, the immune response can be regulated or modulated at a point in time downstream from the activation of the receptor to the signaling pathway (eg, downstream from TLR binding or downstream from TLR activation or recognition). (E.g., increase or decrease bias to Th immune response). Thus, patients also use active agents or combinations of active agents (delivered using the compositions of the invention) that deflect the immune response by acting intracellularly on elements of downstream signaling pathways. Can be treated.

  The present invention induces cellular signaling (eg, activation) of any of the MAP kinase pathways, including the ERK 1/2 pathway, using the desired active agent delivered using the composition of the present invention. Thereby providing a method for biasing against a Th2 immune response. The induced MAP kinase pathway can be characterized by an increased amount of phosphorylated components and / or an extended duration of the MAP kinase pathway, including ERK 1/2.

  In another embodiment of the methods of the invention, an active agent that modulates the ERK 1/2 MAP kinase pathway to regulate a TH2 immune response may be delivered via a composition of the invention. . In this embodiment, by way of example, an agonist of TLR (eg, TLR-2) induces phosphorylation of ERK 1/2 to increase the TH2 immune response. In yet another embodiment of the method of the present invention, the active agent modulates the intracellular c-FOS pathway, thereby regulating the TH2 immune response. Can be delivered via. In this embodiment, by way of example, an agonist of the c-fos pathway induces c-fos expression and / or phosphorylation of c-fos to increase the TH2 immune response. In still further embodiments of the methods of the invention, the active agent modulates a Th2 immune response by acting on elements of TLR2 or downstream signaling pathways (eg, ERK 1/2 MAP kinase pathway and c-FOS pathway). As such, it can be delivered via the composition of the present invention. For example, the active agent delivered via the composition of the invention modulates IL-10 production or activity (eg, increases IL-10 production or upregulates IL-10). Can be used for

  In one embodiment, a method for biasing against a Th2 immune response mediates (eg inhibits) the production of IL12 by administering an active agent delivered via a composition of the invention p38. And / or including diminishing or inhibiting signaling of the JNK pathway, thereby biasing the Th1 response. In another embodiment, a method for biasing against a Th2 immune response mediates IL12 production by administering micelles or particles of the invention comprising a desired active agent (or combination thereof) (eg, Reduce or inhibit the phosphorylated amount of p38 and / or JNK, or bias the duration of phosphorylation of p38 and / or JNK, thereby biasing against the Th1 response Or including inhibiting.

  The present invention also provides for cellular signaling (eg, activation) of any of the MAP kinase pathways, including the p38 and / or JNK pathways, by administering an active agent delivered via a composition of the present invention. A method for biasing against a Th1 immune response is provided. The induced p38 and / or JNK pathway can be characterized by an increase in the amount and / or an increase in duration of the phosphorylated component of the MAP kinase pathway, including p38 and / or JNK.

  In one embodiment, a method for biasing against a Th1 immune response comprises administering an active agent delivered via a composition of the present invention to provide signalin of the ERK 1/2 and / or c-fos pathway. Weakening or inhibiting. In another embodiment, a method for biasing against a Th1 immune response comprises phosphorylating ERK 1/2 and / or c by administering an active agent delivered via a composition of the invention. -Reducing or inhibiting the amount of fos, or shortening or inhibiting the duration of phosphorylation of ERK 1/2 and / or c-fos.

  Furthermore, the present invention provides a method for modulating a TH2 immune response, the method comprising a T cell (eg, a naive T cell) and an ERK 1/2 delivered via the composition of the present invention. Contacting TLR positive cells (eg, DCs) treated in culture containing a TLR agonist (eg, TLR-2 agonist) that activates the pathway and / or activates the c-fos or c-fos pathway Process.

  Furthermore, the present invention provides a method for modulating a TH1 immune response, which method comprises a T cell (eg, a naïve T cell) and a p38 pathway and / or delivered via a composition of the present invention. Or contacting a TLR positive cell treated in a culture comprising a TLR agonist that activates the JNK pathway (eg, a TLR-4 agonist).

  The present invention provides methods for treating a subject having an immune-related condition or disease (eg, allergies, autoimmune diseases, and other immune-related conditions, including cancer) The method comprises administering to the subject an effective amount of any substance of the present invention delivered via a composition of the present invention to bias against or contrary to a Th1, Th2 or Th0 immune response. The process of carrying out is included. The subject can be a cow, pig, mouse, horse, dog, cat, monkey, human, sheep, fish or bird.

  In one embodiment, a subject having a condition or disease associated with a strong Th2 response activates cell signaling in the subject to bias against a Th1 immune response delivered via a composition of the invention. To be treated with the substance of the invention. Diseases characterized by a strong Th2 response include, but are not limited to, allergies, asthma, and chronic obstructive pulmonary disease (COPD (eg emphysema or chronic bronchitis)).

  In another embodiment, a subject having a condition or disease associated with a strong Th2 response is treated with a molecule that inhibits biasing against a Th2 immune response delivered via a composition of the invention. Is done.

  In one embodiment, a subject having a condition or disease associated with a strong Th1 response activates cell signaling in the subject to bias against a Th2 immune response delivered via a composition of the invention. It is treated with the substance of the invention Diseases characterized by a strong Th1 response include, but are not limited to diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, and systemic lupus erythematosus.

  In another embodiment, a subject having a condition or disease associated with a strong Th1 response is used with an active substance that inhibits biasing against a Th1 immune response delivered via a composition of the invention. To be treated.

Advantages of the invention The polyketal particles of the present invention contain a wide range of active substances, from small inhibitors to large proteins, or can encapsulate them, delivering active substances to a subject suffering from a disease or condition Or may be used as a preventive measure.

  In one embodiment, the active agent added to or encapsulated by the polyketal can remain active in the subject for several weeks. In addition, polyketals containing active substances can be lyophilized and stored for several months without degradation. This polyketal has a controllable degradation profile in various pH ranges and can be finely tuned for precise release of the active substance, if necessary.

  The polyketal polymers of the present invention carry the active substance immediately after administration due to, for example, the problems associated with administering the active substance to the area surrounding the cardiomyocytes, i. It should overcome the increased likelihood of being left. Larger, micron-scale polyketals of the present invention should overcome this obstacle and allow polyketals containing or encapsulating active agents to remain in the local microenvironment.

  The polyketals of the present invention have several unique properties that make them ideal as active vehicle delivery vehicles or preventive measures for the treatment of disorders or diseases (eg, treatment of the myocardium): (1) The polyketals of the present invention can be degraded on a time scale, which can be easily manipulated from 1-2 days to several weeks at various pH values; (2) the polyketals of the present invention can be converted to neutral excretable compounds. (3) The polyketals can be solid particles and can be stable over an extended period after formulation; and (3) can be hydrolyzed and thus produce less protein degradation and denaturation than polyester-based microparticles; 4) The properties of the polyketal can be easily manipulated to change the particle size, shape and porosity.

  In one embodiment of the present invention, altering the percentage of the polyketal copolymer to provide a controllable release of the active agent added to or encapsulated by the polyketal. Hydrolysis kinetics can be altered or finely tuned. For example, the monomer composition PK4 provides faster hydrolysis kinetics than PK3 at pH 4.5. Similarly, PK3 has faster hydrolysis kinetics than PK2 or PK5, while PK2 and PK5 have faster hydrolysis kinetics than PK1 or PK6. Thus, the active substance added to PK4 is released faster than the active substance added to PK3; the active substance added to PK3 is released faster than the active substance added to PK2 or PK5; and Active substances added to PK2 or PK5 are released faster than active substances added to PK1 or PK6.

  In another embodiment of the present invention, the polyketals of the present invention can be mixed to alter or fine tune the release rate of the active agent added to or encapsulated by the polyketals. For example, polyketals with fast hydrolysis kinetics (eg, PK4 or PK3) can be mixed with polyketals with slower hydrolysis kinetics (eg, PK1 or PK6) and co-administered to a subject. The active ingredient added to PK4 or PK3 is rapidly released in the subject, providing the subject with an immediate release (IR) dose of the active agent. On the other hand, the active substance added to PK1 or PK6 is released at a slower rate, allowing a gradual or extended release (ER) of the active substance in the subject.

  Another advantage of the present invention is that in one embodiment, a linker can be attached to the polyketal polymer. This polyketal polymer can then be used to form particles with linkers. A particle with a linker allows for the delivery of one or more active agents (ie, through encapsulation of the active agent by the particle and attachment of the active agent-particle to the linker) to the subject. In one embodiment, this dual mode of active agent delivery provides a double release time for the active agent, where the active agent attached to the linker releases faster than the encapsulated active agent. Is done.

  The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are in no way intended to limit the scope of the invention otherwise.

Example 1
Synthesis of cross-linked micelles containing antigen Cross-linked micelles containing the protein antigen ovalbumin and immunostimulatory DNA were synthesized in a two-step process. Initially, micelles were formed between the cationic block copolymer PEG-polylysine-thiopyridal and negatively charged FITC-ovalbumin (FITC-OVA) and immunostimulatory DNA (ISS-DNA). These micelles contained 10 mg / ml PEG-polylysine-thiopyridal, 0.5 mg / ml FITC-OVA, and 0.5 mg / ml ISS-DNA. The micelles were allowed to form for 1 hour and then the micelles were crosslinked with 0.4 mg / ml dithio-ethylene glycol. The cross-linking reaction is described in U.S. Pat. V. Monitoring by activity (342 nm) showed that the thiopyridal group reacted quantitatively at room temperature after 1 hour. The micelles were centrifuged by 100 kD spin-filter (centricon), and the recovered solution was analyzed for FITC fluorescence (excitation 494 nm, emission 510 nm) to determine the encapsulation efficiency of FITC-OVA in the micelles. This indicated that more than 95% of FITC-OVA was encapsulated in the micelles.

(Example 2)
Synthesis of polyketal particles (single emulsion method for delivery of hydrophobic drugs)
The particles were synthesized with poly (l, 4-phenylene-acetone dimethylene ketal) using an oil-in-water emulsion method. Briefly, 10 mg 2.1 mg ERK inhibitor UO126 and 0.1 g chloro-methyl fluorescein diacetate (CMFDA) were dissolved in 0.5 mL CHCl ₃ (containing 0.1% triethylamine). . This solution was then added to 5 mL of pH 9 buffer (10 mM NaHCO ₃ ) containing 2 mg / ml polyvinyl alcohol (PVA, 31-50 kDa, Aldrich). The oil-water mixture was shaken briefly and then sonicated (Branson Sonifier 250) for 2-3 minutes at 40 Watts to form a fine oil / water emulsion. The emulsion was stirred under N ₂ flow for at least 3 hours to evaporate the solvent and produce a particle suspension. Particle size was analyzed by dynamic light scattering (DLS) and showed an average diameter of 282 nm.

(Example 3)
Synthesis of polyketal particles encapsulating ovalbumin (double emulsion method for the synthesis of hydrophilic drugs)
Synthesis of polyketal particles Polyketal particles (PKN) containing FITC-ovalbumin were made using a double emulsion method. First, 20 mg of poly (1,4-phenylene acetone dimethylene ketal) (PPADK) dissolved in 500 μL of chloroform was added to 100 μL of FITC-Ova solution (about 0.7 mg). The mixture was sonicated at 40 watts for 1 minute to form a primary emulsion. Next, 5 mL of 0.2% w / v polyvinyl alcohol (PVA, Aldrich) in 10 mM pH 9 sodium phosphate buffer was added and the mixture was sonicated at 40 watts for at least 1 min. The next emulsion was formed. The emulsion was mixed for 4 hours under nitrogen bubbling, after which the volume was made up to 5 mL with buffer. Two batches of FITC-Ova containing PKN were prepared in the same manner as two batches of plain PKN (without FITC-Ova). The PKN suspension was stored at 4 ° C.

  Particle sizing was determined by dynamic light scattering (DLS). Two batches of FITC-ovalbumin loaded PKN had effective diameters of 426 nm and 462 nm, and the empty PKN batches were 321 nm and 347 nm.

FITC-Ova labeling Chicken egg albumin (ovalbumin) was labeled with fluorescein as follows. Ovalbumin was dissolved in 200 mM pH 9 NaHCO ₃ buffer at 10 mg / mL. Fluorescein isothiocyanate (FITC) was dissolved in DMSO at 10 mg / mL. Next, 2.5 mL of ovalbumin solution (25 mg, 0.56 mmol) was mixed with 50 μL of FITC / DMSO (0.5 mg, 1.28 mmol) for at least 1 hour at 30 ° C. The product was filtered through a Sephadex PD-10 column to remove free dye; the column was loaded with 2.5 mL of product and eluted with 2.5 mL of water. The concentration of the obtained ovalbumin was about 7 mg / mL. The degree of labeling was calculated to be 1.09 by measuring the absorption of FITC at 497 nm and using the estimated concentration of ovalbumin.

Determination of FITC-Ova Encapsulation in PKN Two FITC-Ova PKN batches were diluted with 5-fold water, and a portion of each diluted sample was filtered through a 0.1 μm Super syringe filter (Pall Acrodisc). . These filtered and unfiltered samples were further diluted in 10 × pH 9 sodium phosphate buffer and fluorescence was measured at an excitation wavelength of 494 nm and an emission wavelength of 520 nm (FIG. 1).

  The encapsulation efficiency was calculated using the fluorescence intensity of the filtered and unfiltered samples as follows:

Example 4 Synthesis and Decomposition of Ketal Skeleton Polymer (Polyketal) FIG. 2 shows the following: (A) 1,4-benzenedimethanol and 2,2-dimethoxy to produce ketal intermediate 1 Ketal exchange reaction with propane. (B) One step polymerization to produce polyketal 2. Reaction steps A and B proceed by distilling the by-product methanol. (C) Formation of drug loaded particles by solvent evaporation method. The particles represent a pH sensitive degradation into low molecular weight effluxable compounds.

Synthesis The polyketals are synthesized by a novel polymerization strategy based on the ketal exchange reaction (Lorette, NB; Howard, WL J. Org. Chem. 1960, 25, 521-525). This reaction is generally used to introduce protecting groups into low molecular weight alcohols and has not previously been used to synthesize polymers. However, the inventors now note that the ketal exchange reaction can be used to synthesize acid-sensitive polymers by simply reacting 2,2-dimethoxypropane (DMP) with a diol. To clarify. We propose that the above polymerization occurs by the reaction mechanism of FIG. The ketal exchange reaction is an equilibrium reaction involving protonation of DMP and subsequent nucleophilic attack by alcohol. This equilibrium is shifted relative to the formation of the ketal intermediate 1 by distilling off the byproduct methanol. As this reaction proceeds, one molecule binds in a stepwise fashion to form polyketal 2.

Polyketal 2 was obtained in 48% yield by a typical polymerization of DMP and 1,4-benzenedimethanol (BDM). The polymerization was carried out in a 25 mL two-necked flask connected to a short path distillation head. BDM (1.0 g, 7.3 mmol, Aldrich) dissolved in 10 mL warm ethyl acetate was added to 10 mL distilled benzene maintained at 100 ° C. Then recrystallized p-toluenesulfonic acid (5.5 mg, 0.029 mmol, Aldrich) dissolved in 550 μL of ethyl acetate was added. After distilling off ethyl acetate, distilled DMP (900 μL, 7.4 mmol, Aldrich) was added to start the reaction. An additional volume of DMP (each volume consisting of 2 mL of benzene and 300-500 μL of DMP) was added via a metering funnel. Each volume was added over a period of 30-40 minutes with an interphase of 30 minutes in between. The total duration of the reaction was 7 hours. The reaction was stopped by the addition of 100 μL triethylamine and precipitated into cold hexane. The crude product was vacuum filtered, rinsed with ether and hexane, and dried in vacuo to give 600 mg of white solid product (48% yield). The recovered polymer was analyzed by GPC and ¹ H NMR.

FIG. 3A shows a GPC trace from one batch. In this batch, a polydispersity index of Mw = 4000 and 1.54 was obtained for a degree of polymerization of 22.5 repeat units. ^{The 1} H NMR spectrum (FIG. 3B) confirms that the 2 repeat units contain a dimethyl ketal group (“6a”). Together, GPC and ¹ H NMR data provide evidence for successful polyketal 2 synthesis.

Hydrolysis The hydrolysis kinetics of 2 were measured at pH values for lysosomes (pH 5.0) and blood flow (pH 7.4). Grind Polyketal 2 into a fine powder and add this powder to a solution containing deuterium at pH 7.4 (phosphate buffer), pH 5.0 (acetate buffer), and pH 1.0 (DCl) The hydrolysis rate was measured. The suspension was stirred at 37 ° C. and data points were taken at 3 hours, 24 hours, 48 hours, and 72 hours. Each suspension was centrifuged for 4 minutes at 1800 g and the supernatant was analyzed by ¹ H NMR. These spectra contained peaks for BDM (7.24 ppm and 4.47 ppm) and peaks for acetone (2.05 ppm). The average of the integration of the two BDM peaks was used to determine the relative degree of hydrolysis. The percent hydrolysis was calculated by dividing the average of the BDM peak of the pH 7.4 or 5.0 sample by the pH 1.0 versus the average of the BDM peak of the control batch.

  The exponential decay half-life was calculated as 102 hours at pH 7.4 and 35 hours at pH 5.0. This represents a 3-fold rate increase from pH 7.4 to 5.0 (FIG. 4). The pH sensitivity of 2 is described in Kwon et al., (Kwon, YJ; Standley, SM; Goodwin, AP; Gillies, ER; Frechet, JM Mol. Pharm. 2005, 2, 83-91) significantly less than the pH sensitivity for water-soluble ketals. We assume that a smaller pH sensitivity of 2 is due to its water insolubility. This water insolubility creates another rate limiting step that limits the diffusion of water and is insensitive to pH. The kinetics of the diffusion of water into the material made from 2 depends on the size of this particle, and we predict that smaller particles have greater pH sensitivity than ground particles. .

(Example 5)
Synthesis of micron-sized particles Polyketal 2 (Example 4, from above) was also used to synthesize micron-sized particles. Oil-in-water emulsion method (Panyam, J .; Williams, D .; Dash, A .; Leslie-Pelecky, D .; Labhasetwar, V. J. Pharm. Sci. 2004, 93, 1804-1814) Was used to form. Briefly, 50 mg of 2 dissolved in 1 mL of CHCl ₃ (containing 0.1% triethylamine) is added to 5 mL of 10 mM containing various amounts of polyvinyl alcohol (PVA, 31-50 kDa, Aldrich) as an emulsifier. Added to NaHCO ₃ pH 9 buffer. The oil-water mixture was shaken briefly and then sonicated (Branson Sonifier 250) for 2-3 minutes at 40 Watts to form a fine oil / water emulsion. The emulsion was stirred under N ₂ flow for at least 3 hours to evaporate the solvent and produce a particle suspension.

  Particle size was analyzed by dynamic light scattering (DLS) and SEM. A sample of DLS was prepared by diluting the particle suspension in 10 mL pH 9 buffer and stabilizing the larger particles. An aliquot from the liquid portion of each vial was then diluted for DLS particle sizing (Brookhaven 90Plus particle sizer). The particle suspension was centrifuged for 10 minutes (5000 g, 4 ° C.), washed with distilled water, and the recovered pellet was lyophilized to give a SEM sample a PVA: polyketal ratio of 0.2. : 1.

  As expected, the particle size was sensitive to the ratio of PVA to polyketal. The diameter of the DLS particles was 520 nm, 290 nm, and 280 nm for samples containing PVA: polyketal ratios of 0.2: 1, 0.8: 1, and 2: 1, respectively. The 0.2: 1 batch SEM images (FIGS. 5A and 5B) confirm that the polyketals form micron-sized particles with a particle size distribution ranging from 0.5-30 μm in diameter.

(Example 6)
Synthesis and Characterization of Micelles and Polyketal Particles Materials and Methods Encapsulation of Dexamethasone in Polyketal Particles The anti-inflammatory drug dexamethasone (Dex, Sigma) was encapsulated in particles made using the polyketal 2 described above. The particles loaded with Dex were formulated using the same procedure as above except that the oil phase contained Dex at a concentration of 5 mg / ml and a 1: 1 PVA: polyketal ratio was used. . SEM images of these particles reveal that these particles are 200-600 nm in diameter (FIG. 5C). Sizing the particles with DLS showed an effective diameter of 250 nm for a batch of particles loaded with Dex. The Dex encapsulation efficiency ranged between 43-53%. Control batches were prepared using polyketal / PVA alone and Dex alone. To measure Dex encapsulation, each particle batch was resuspended in pH 9 buffer and then the aliquot was further diluted. A portion was filtered through a 0.1 [mu] m Super membrane Acrodisc syringe filter (Pall Corp.) and the absorbance of the filtrate at 242 nm was recorded by a Shimadzu UV-1700 spectrophotometer. The encapsulation efficiency was calculated as (A _Dex -A _DexPoly ) / (A _Dex -A _Poly ). Here, A is the absorbance at 242 nm, and the subscripts “Poly”, “Dex”, and “DexPoly” refer to “polyketal only”, “Dex only”, and “Dex + polyketal” samples, respectively. These calculations yielded 43% to 53% Dex encapsulation efficiency for various samples.

Micelle Formation FIG. 8 is a schematic showing the design and synthesis of peptide-crosslinked micelles. Step 1: ISS DNA and I are mixed to form micelles (non-crosslinked micelles). Step 2: These micelles are then cross-linked with antigenic peptide (II) to produce a delivery system that can encapsulate both immunostimulatory molecules and peptide antigens. After phagocytosis by APC, peptide-crosslinked micelles release their constituents.

  The HIV peptide vaccine was synthesized using a PCM strategy with the peptide CGCRIQRGPGRAVVTIGKCGCG (II). The peptide II is derived from the GP-120 protein and contains the sequence RIQRGPGRAFVTIGK, which is both a class I antigen and a class II antigen. First, 0.5 mg of I is mixed with 0.1 mg of ISS DNA (5-TCCATGACGTTCCTGACGTTT-3) (charge ratio was 1 to 15 (− / +)) in 0.5 ml of 50 mM PBS. Thus, micelles were formed between I and ISS-DNA. The dynamic light scattering of these micelles using the Cumulants method showed that these micelles had an average diameter of 57.0 nm. The micelles were then cross-linked by adding 0.1 mg of II to the micelles (the thiopyridal group in cysteine to I in an equimolar ratio of II). The peptide II was incorporated into the micelle by a disulfide exchange reaction.

FIG. 9 shows the synthesis and characterization of PEG-polylysine thiopyridal. A. Synthesis of PEG-polylysine thiopyridal (I). 44 micromolar PEG-poly-1-lysine (containing PEG = 5 kd and poly-1-lysine = 5,000) was dissolved in 1 ml DMF in a 5 ml round bottom flask equipped with a stir bar ( In order to completely dissolve the polymer, overnight stirring at room temperature was required). 415 micromoles of hydroxyl-ethyl thiopyridal acrylate and 58 μl of triethylamine were then added to the PEG-poly-1-lysine solution and the reaction was allowed to continue for 24 hours at room temperature. The product was isolated by precipitating the reaction solution in 15 ml of ice-cold diethyl ether. This yield was 88.2%. B. ¹ H-NMR spectrum of PEG-PLL-thiopyridal in D ₂ O. The product of (A) was analyzed by ¹ H NMR in D ₂ O. Proton of pyridine (—NC ₅ H ₄ : δ = 7.101 ppm, 7.629 ppm, 8.187 ppm) vs. poly-1-lysine α, β, γ-methylene protons (—CH ₂ CH ₂ CH ₂ : δ) = 1.122 ppm, 1.285 ppm, 1.553 ppm) by comparing the ratio of peak intensities to determine the percentage of alkylated amine. This indicated that 100% of the amine had reacted. C. / D. Dynamic light scattering analysis of uncrosslinked PCM (C) and peptide crosslinked PCM (D). A pH 7.4 50 mM PBS buffer solution containing 0.06 mg / ml PEG-polylysine thiopyridal and 20 μg / ml ISS DNA was prepared and filtered through a 200 nm syringe filter. The solution was then analyzed by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments) using the Cumulant method (C). The solution was then cross-linked by adding 0.12 mg peptide antigen (II), and after 3 hours of reaction, the solution was analyzed by DLS as described above (cross-linked micelle size and size). Is shown in D). E. Cross-linking reaction of cysteine in peptide antigen (II). F. UV analysis for cross-linking reaction between peptide antigen (II) and block copolymer micelles. In an Eppendorf tube, mix 0.5 mg I with 0.1 mg ISS DNA in 0.5 ml 50 mM NaH ₂ PO ₄ buffer (pH 7.4) (amine to phosphate ratio 15/1). Block copolymer micelles were formed between I and ISS DNA. After 2 hours incubation at room temperature, 0.1 mg II (representing a 1: 1 cysteine: thiopyridal ratio) was added to the micelles. The cross-linking reaction between cysteines in II with thiopyridal groups in the micelles was determined by UV analysis at 342 nm (representing released thiopyridone). The percentage of reacted cysteine groups was determined by the following formula:

Where: ABS ₁ = UV absorption at 342 nm for the peptide-crosslinked micelle reaction (black circle); ABS ₂ = UV absorption at 342 nm for uncrosslinked micelle without peptide (white square); ABS ₀ = UV absorption at 342 nm (open circles) when all thiopyridone groups have reacted (by addition of DTT).

  FIG. 10 shows the effect of GSH on peptide and DNA release. A. GSH sensitive peptide release. To determine whether PCM releases peptide antigens after phagocytosis, the stimulated reactive release of peptides from PCM due to the presence of GSH was examined. PCM was incubated with different concentrations of GSH for 24 hours in 50 mM pH 7.4 PBS buffer and then analyzed by HPLC to determine peptide release. This figure reveals that the release of the peptide is caused by the presence of GSH. Incubation of PCM with 10 mM GSH (intracellular level) induces 71% release of the peptide, whereas incubation of PCM with buffer only causes only 10% release of the peptide. B. GSH sensitive DNA release. The ability of PCM to protect encapsulated ISS-DNA from degradation by serum nucleases was examined. PCMs were synthesized (as described above) and incubated with 10% serum for 12 hours, then these PCMs were examined by gel electrophoresis to determine the stability of the encapsulated ISS-DNA. It was. As a control, ISS-DNA itself was incubated with serum. This figure shows that ISS-DNA itself is fully hydrolyzed in 10% serum (lane 2), in contrast, ISS-DNA encapsulated in PCM probably has nucleases entering the micelles. Evidence that it is protected from serum nucleases due to the effect of cross-linking that must prevent (lane 3). C. ISS-DNA is protected from serum nucleases in PCM. An important advantage of the PCM strategy is to generate a cross-linked delivery system. This cross-linking stabilizes PCM in vivo. To determine the stability of PCM against degradation by mixing the negatively charged polymer, poly (vinyl sulfate) (PVS) with PCM, and then to determine the amount of ISS-DNA displaced by PVS, This mixture was analyzed by gel electrophoresis. As a control, PVS was also incubated with uncrosslinked micelles composed solely of II and ISS-DNA. Figure A, lane 4 demonstrates that PVS can destroy uncrosslinked micelles composed solely of ISS-DNA and II. In contrast, A, lane 5, shows that PVS displaces PCM-derived ISS-DNA, possibly due to peptide cross-linking preventing PVS from dispersing in the micelles and replacing ISS-DNA. Make it clear that you can't. Importantly, after incubation of PCM with intracellular concentrations of GSH, PCM releases encapsulated ISS-DNA in the presence of PVS. This reveals that the micelles release their contents after phagocytosis (lanes 2 and 3 of b)). The charge ratio of ISS-DNA to I is 1 to 15 (− / +); 1 μg of DNA was loaded into each lane; to induce release of encapsulated ISS-DNA, GSH (100 μM ) Was added to lane 3. All samples were incubated with 10% serum for 12 hours at room temperature.

  FIG. 11 shows block copolymer micelles: Chemical structure of PEG-poly (lysine-thio-pyridyl). The PEG chain provides stability to the micelles and the polylysine segment is used for electrostatic interactions with proteins and DNA or RNA. The thiopyridal group is due to cross-linking through the resulting disulfide bond. B. Step I: PEG-poly (lysine-thio-pyridal) is mixed with DNA and protein to form micelles with PEG on the outside. C. Step II: Crosslink micelles with dithiol-containing molecules (eg, di-thioethylene glycol). D. Cross-linked micelles are reduced with glutathione, which has a higher concentration in cells than in blood.

FIG. 12 shows micelle immunology. A. Uptake of SIINFEKL-CFSE micelles by DCs derived from human monocytes. DCs derived from human PBMC (cultured on day 6) were treated with SIINFEKL / CFSE micelles at a concentration of 10 μg / ml, 5 × 10 ^{* 5} cells / well, 96-U well, RPMI / 10% FCS at 37 ° C. for 4 hours. Used to pulse. Cells were washed, fixed in this form, and prepared for confocal microscopy imaging. B. Efficient uptake of micelles encapsulating SIINFEKL peptides by DCs derived from human monocytes. Human PBMC-derived DCs (culture day 6) were simply plain SIINFEKL at 37 ° C. for 4 hours at concentrations of 10 μg / ml, 5 × 10 ^{* 5} cells / well, 96-U well, RPMI / 10% FCS. Pulsed with micelles (2, 3) formulated with / CFSE (1) or SIINFEKL / CFSE. Cells were washed and stained for CD11c and HLADR. Cells were gated on CD11c + cells, HLADR + cells and analyzed for CFSE fluorescence. C. Efficient uptake of micelles encapsulated SIINFEKL peptide by mouse DCs and macrophages. Whole mouse C57B1 / 6J FLT3-L spleen cells were simply labeled with 1 or 10 μg / ml CFSE at 37 ° C. at a concentration of 1 × 10 ^{* 6} cells / well, 96-U well, RPMI / 10% FCS. SIINFEKL (1) or peptide (2,3) were pulsed with micelles formulated (blue line) for 4 hours or left untreated (red line). Cells were stained for CD11c and CD11b and analyzed for CFSE positive fluorescence.

FIG. 13 is a bar graph showing that micelles formulated with SIINFEKL peptide induce a strong T cell response in vitro. Syngeneic ^{C57B1 / 6J H-2 b FLT3} -L from treated mice of purified CD11c ^+, pulsed for 4 hours, the indicated concentrations and combinations micelles formulated mere SIINFEKL peptide or SIINFEKL peptide did. Cells were washed and cultured with total OT-1 spleen cells for 20 hours (left panel) or 96 hours (right panel). Cells were stained for CD8 and intracellular IFNγ and analyzed with a flow cytometer. The graph shows that micelle SIINFEKL 213.3 (black bar), micelle SIINFEKL 213.2 (grey bar), simple SIINFEKL (white bar), medium (dot bar) stimulated CD11c + DC.

FIG. 14 shows micelle immunology. A. Efficient uptake of micelles encapsulating OVA protein by mouse DCs and macrophages. Whole mouse C57B1 / 6J FLT3-L spleen cells were labeled with 1 or 10 μg / ml FITC at 37 ° C., concentration 1 × 10 ^{* 6} cells / well, 96-U well, RPMI / 10% FCS, Ovalbumin (1) or micelles formulated with protein (2) were pulsed for 1 hour (black line), 5 hours (dotted line), or left untreated (shaded). Cells were stained for CD11c and CD11b and analyzed for FITC positive fluorescence. B. OVA / CpG encapsulated micelles activate DCs in vitro. Whole mouse C57B1 / 6J FLT3-L spleen cells with 1 μg / ml of simple OVA (1), simple OVA + 1 μg of CpG (2) or micelle formulated with OVA (3) or micelle OVA + 1 μg of CpG (4) Pulsed for 24 hours. CD11c + cells were analyzed for CD80 or CD86 markers. Data represent isotype controls (shaded), untreated cells (gray line) or stimulated cells (black line).

FIG. 15 is a graph showing the immunology of polyketal particles. Incorporation of U0126 encapsulated polyketal particles (PKN) by mouse DCs and macrophages in vitro. Total mouse C57B1 / 6J FLT3-L spleen cells were treated with 1 × 10 ^{* 6} cells / well, 96-U well, RPMI / 10% FCS at 37 ° C. for 5 hours, 1 or 10 μg / Pulsed with polyketal particles labeled with ml CMFDA. Cells were stained for CD11c and CD11b and analyzed for CMFDA positive fluorescence. Data represent cells treated in culture (shaded) or 5-hour uptake (black line).

  FIG. 16 is a schematic diagram showing an experimental outline of in vitro T cell stimulation using the OVA-OT / 1 transgenic model.

FIG. 17 shows micelle immunology. A. Antigen-formulated micelles induce a strong T cell response in vitro. Syngeneic C57B1 / 6J ^{H-2 b} mice of purified CD11c ⁺ cells for 4 hours, pulsed with the concentration and ovalbumin are shown a combination of just CpG or formulated micelles. Cells were washed and 1 × 10 ^{* 5} CD11c + were cultured for 5 days with 1 × 10 ^{* 6} total OT-1 (SIINFEKL specific) spleen cells. After 5 days, cells were restimulated with SIINFEKL peptide (1 μg / ml) with BFA (5 μg / ml) for 6 hours and stained for CD8 and intracellular IFNγ. The left panel represents a flow cytometer analysis; the right panel summarizes these data. B. Antigen-formulated micelles overcome the CD4 + -dependent CD8 + T cell induction mechanism. Syngeneic C57B1 / 6J ^{H-2 b} mice of purified CD11c ⁺ cells, and the indicated concentrations and ovalbumin using a combination of just CpG or prescribed micelle was 4 hours pulse. Cells were washed and 1 × 10 ^{* 5} CD11c was cultured with 5 × 10 ^{* 5} CD8 + MACS purified OT-1 spleen cells (approximately 90% purity) for 5 days. After 5 days, cells were restimulated with SIINFEKL peptide (1 μg / ml) with BFA (5 μg / ml) for 6 hours and stained for CD8 and intracellular IFNγ. The left panel represents a flow cytometer analysis; the right panel summarizes these data. C. Antigen formulated micelles activate DCs in vivo. C57B1 / 6J mice (2 / group) received micelles formulated in 5 μg / mouse of simple ovalbumin / CpG or 500 μl PBS i. v. Injected. Spleens were removed 4 and 24 hours after injection, treated with collagenase (30 min / 37 ° C.), homogenized, and treated with erythrocyte lysis buffer. CD11c + cells were stained for CD80 or CD86 markers and analyzed using a flow cytometer. Data represent isotype controls (shaded), untreated mice (gray line) or mice injected with antigen (black line).

FIG. 18 shows the immunology of micelles and polyketal particles. A. Vaccinated micelles induce a strong T cell response in vivo—CD8 ⁺ IFNγ ⁺ . A cohort of 4 C57B1 / 6J mice received 5 μg / mouse of OVA (1) or OVA + CpG (2), micelle OVA (3), micelle OVA / CpG (4) micelles. c. Vaccinated with. Using the same antigen formulation, animals were boosted on day 36 (boost 1) and day 84 (boost 2). Blood was collected 6 days after boost 2, PBMCs were isolated using the Histopaque gradient method, restimulated with SIINFEKL peptide (1 μg / ml) and BFA (5 μg / ml) for 6 hours, and CD8 and Stained for intracellular IFNγ. The left panel represents flow cytometry analysis and gating strategy for selected mice; the right panel summarizes these data as% of CD8 ⁺ IFNγ ⁺ cells out of total CD8 ⁺ T cells. B. Vaccinated micelles induce a strong T cell response -CD8 ⁺ TNFα ⁺ in vivo. A cohort of 4 C57B1 / 6J mice received 5 μg / mouse OVA (1) or OVA + CpG (2), micelle OVA (3), micelle OVA / CpG (4), s. c. Vaccinated with. Using the same antigen formulation, animals were boosted on day 36 (boost 1) and day 84 (boost 2). On day 6 after boost 2, blood was collected and PBMC was isolated using the Histopaque gradient method. PBMCs from 4 mice were pooled, restimulated with SIINFEKL peptide (1 μg / ml) and BFA (5 μg / ml) for 6 hours, and stained for CD8 and intracellular TNFα and IL-10. The left panel represents flow cytometry analysis and gating strategy for selected mice; the right panel summarizes these data as% of total CD8 ⁺ T cells, CD8 ⁺ TNFα ⁺ cells. C. Kinetics of specific CD8 + / IFNγ + T cells after OVA / CpG vaccination. A cohort of 4 C57B1 / 6J mice received 5 μg / mouse OVA + CpG (gray line) and micelle OVA / CpG (blue line) s. c. Vaccinated with. Using the same antigen formulation, animals were first immunized on day 0 and boosted on day 36 (boost 1) and day 84 (boost 2). Blood was collected on different days after the first immunization and boost and PBMCs were isolated using the Histopaque gradient method. PBMCs from 4 mice were restimulated with SIINFEKL peptide (1 μg / ml) and BFA (5 μg / ml) for 6 hours and stained for CD8 and intracellular IFNγ. The panel summarizes these data as% of CD8 ⁺ IFNγ ⁺ cells (including SEM error bars) of total CD8 ⁺ T cells. D. Kinetics of specific CD8 + / IFNγ + T cells after OVA + UO126 PKN vaccination. A cohort of 4 C57B1 / 6J mice received 5 μg / mouse OVA + CpG (grey line), micelle OVA / CpG (blue line) and micelle OVA + 10 μg UO126 PKN (red line). c. Vaccinated with. Using the same antigen formulation, animals were first immunized on day 0 and boosted on day 36 (boost 1) and day 84 (boost 2). Blood was collected on different days after the first immunization and boost and PBMCs were isolated using the Histopaque gradient method. PBMCs from 4 mice were restimulated with SIINFEKL peptide (1 μg / ml) and BFA (5 μg / ml) for 6 hours and stained for CD8 and intracellular IFNγ. The panel summarizes these data as% of CD8 ⁺ IFNγ ⁺ cells (including SEM error bars) of total CD8 ⁺ T cells. E. Micelles formulated with vaccines induce strong antigen-specific IgG antibody responses in vivo. Cohorts of 4 C57B1 / 6J mice were included in 5 μg / mouse OVA (1) or OVA + CpG (2), micelle OVA (3), micelle OVA / CpG (4) micelle OVA + 10 μg PKN U0126 (5) or micelle OVA / CpG + 10 μg PKN U0126 (6) was s. c. Vaccinated with. Animals were boosted on days 36 and 84 using the same antigen formulation. On the 4th week after the first immunization (first time), 6 weeks after the first boost (boost 1) and 7 weeks after the second boost (boost 2), phlebotomy was performed. Sera from individual mice were pooled and tested for reactivity of OVA-specific total IgG, IgG1, IgG2a and IgG2b antibodies using plate ELISA. Antibody reactivity was measured as absorbance at 450 nm for serial dilutions of serum. Data are expressed as the reciprocal of specific anti-OVA antibody. F. Vaccinated micelles induce antigen-specific IgE and IgM antibody responses in vivo. Cohorts of 4 C57B1 / 6J mice were included in 5 μg / mouse OVA (1) or OVA + CpG (2), micelle OVA (3), micelle OVA / CpG (4) micelle OVA + 10 μg PKN U0126 (5) or micelle OVA / CpG + 10 μg PKN U0126 (6) was s. c. Vaccinated with. Animals were boosted on days 36 and 84 with the same antigen formulation. Seven weeks after the second boost, phlebotomy was performed. Sera from individual mice were pooled and tested for OVA-specific IgE and IgM antibody reactivity using plate ELISA. Antibody reactivity was measured as the absorbance at 450 nm for serial dilutions of serum. Data are expressed as the reciprocal of specific anti-OVA antibody.

Polyketal PCADK Characterization FIG. 19 shows a polyketal derived from cyclohexane dimethanol. A. Polyketals derived from cyclohexane dimethanol (called PCADK) decompose into cyclohexane dimethanol and acetone, both of which have FDA approval for human use. B. PCADK degrades in an acid sensitive manner. Ketal bonds in PCADK hydrolyze weekly under physiological pH conditions. Hydrolysis of the ketal bond in PCADK was measured by H-NMR at pH of 4.5 and 7.4. At phagosome pH 4.5, the ketal bond of PCADK is hydrolyzed about 30% after 10 days. Based on this result, we expect that CAT-PKN will be fully hydrolyzed within 4-5 weeks after phagocytosis by macrophages.

  FIG. 20 shows an SEM image showing that PCADK-derived particles can encapsulate hydrophobic compounds and drugs such as rhodamine red and ebselen.

  FIG. 21 shows a line graph showing that the release of rhodamine red from PCADK is pH sensitive.

Synthesis and Characterization of Polyketals and Their Particles FIG. 6 is a schematic diagram showing the steps of particle formation. A. Step 1: Dissolve polyketal and drug in chloroform; dissolve polyvinyl alcohol in water. B. Step 2: Add chloroform solution to water and sonicate to produce micron sized droplets. Step 3: Evaporate chloroform to produce particles.

  FIG. 7 shows that polyketal particles loaded with fluorescein are taken up by the liver. Mouse liver tissue section showing release of fluorescein from PKN after intravenous injection.

  FIG. 22 is a chemical notation that a polyketal containing almost any aliphatic diol can be made. The hydrophobicity of the polyketal determines its hydrolysis kinetics.

  FIG. 23 is a photograph showing that FITC-labeled polyketals are phagocytosed by macrophages in the liver. Phagocytosis of PKN by Kupffer cells in vivo. Mice were injected with either FITC-PKN or empty PKN. The livers of these mice were analyzed by histology. Left: FITC-PKN is abundant in Kupffer cells, as evidenced by scattered green fluorescence (magnification 100 ×). Center: Empty PKN produces little background green fluorescence (100 × magnification). Right: Immunohistochemistry (IHC) for FITC (red) confirms uptake by Kupffer cells (magnification 400 ×).

  FIG. A. Double emulsion procedure used to encapsulate catalase and superoxide dismutase in polyketal particles. Using a homogenizer, 50 μL of an aqueous solution of catalase (1 mg / ml) was dispersed in an organic phase consisting of 75 mg of PCADK dissolved in 1 mL of dichloromethane to produce a water-in-oil (w / o) emulsion. The w / o emulsion was then dropped into 25 mL of 4% PVA solution and the solution was mechanically stirred using a homogenizer. The resulting w / o / w emulsion was then poured into 225 mL of 4% PVA solution and mechanically stirred for several hours until the methylene chloride evaporated. The resulting particles were isolated by centrifugation, lyophilized and examined by SEM (B). The encapsulation efficiency of this protein was 35%. These CAT-PKNs have an average diameter of about 8 microns. B. SEM image of catalase-containing particles and fluorescence microscope image of catalase-containing particles. C. Catalase particles have enzymatic activity, as evidenced by the ability to reduce the absorbance of hydrogen peroxide at 240 nm.

References for Examples 4-6:
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Walter, E .; Dreher, D .; Kok, M .; Thiele, L .; Kiama, S .; G. Gehr, P .; Merkle, H .; P. J. et al. Controlled Release 2001, 76, 149-168.
van Apeldoorn, A.M. A. Van Manen, H .; -J. Bezemer, J .; M.M. De Bruijn, J .; D. Van Blitterswijk, C .; A. Otto, C .; J. et al. Am. Chem. Soc. 2004, 126, 13226-13227.
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Example 7A: Synthesis and characterization of PK particles for the treatment of myocardial infarction In this example, the use of polyketal (PK) particles for the delivery of small molecule inhibitors to the myocardium is described. The PK is designed to deliver therapeutic agents to the heart and treat acute myocardial infarction. PK is formulated from a new class of polymers, polyketals, which contain ketal linkages in their backbone (FIG. 30).

Materials and Methods Synthesis of poly (cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK) PCADK was synthesized in a 50 mL two-necked flask connected to a short-path distilling head. First, 5.5 mg of recrystallized p-toluenesulfonic acid (0.029 mmol, Aldrich) dissolved in 6.82 mL of ethyl acetate was added to 1,4-cyclohexanedimethanol (12.98 g, 90 To a 30 mL benzene solution (held at 100 ° C.) containing 0.0 mmol, Aldrich). The ethyl acetate was evaporated and distilled 2,2-dimethoxypropane (10.94 mL, 90.0 mmol, Aldrich) was added to the benzene solution to initiate the polymerization reaction. Next, additional doses of 2,2-dimethoxypropane (5 mL) and benzene (25 mL) were added to the reaction via a metering funnel every hour over a 6 hour period, and 2,2-dimethoxy distilled off. Propane and benzene were offset. After 8 hours, the reaction was stopped by adding 500 μL of triethylamine. The polymer was isolated by precipitation with cold hexane (stored at −20 ° C.) followed by vacuum filtration. The molecular weight of PCADK was determined by gel permeation chromatography (GPC) (Shimadzu) equipped with a UV detector as shown in FIG. THF was used as the mobile phase at a flow rate of 1 mL / min. Polystyrene standards from Polymer Laboratories (Amherst, Mass.) Were used to establish a molecular weight calibration curve. This compound was used to generate PCADK particles in all subsequent experiments.

Particles can be produced using SB239063 added to PCADK SB239063 is an inhibitor of p38. FIG. 29 is a schematic diagram of p38 activation, including upstream effector activation and activation results.

  An experimental protocol was developed based on the procedure used to formulate SOD into PLGA-based microparticles (Hayashi et al., Entrapment of proteins in poly (L-lactide) microspheres using reversed mirevent evelopment 11). 2): 337-40). Briefly, a 500 μL solution of SB239063 (1 mg / mL) was added to 1.0 mL of methylene chloride containing 75 mg of PCADK. Next, this mixture was dropped into 5 mL of 4% (w / v) polyvinyl alcohol (PVA) aqueous solution, and stirred at 6,000 rpm for 1 minute using a homogenizer. The resulting w / o emulsion was then poured into 25 mL of pH 7.4 buffer and the mixture was stirred for several hours to evaporate the methylene chloride. The resulting particles were isolated by centrifugation and lyophilized to produce a white solid powder. Using a standard curve for SB239063 only, U.S. at 320 nm. V. The protein encapsulation efficiency of SB293063 microparticles, determined by absorbance, was 25-50%. The SEM images of SB239063-PCADK particles shown in FIG. 32 demonstrate that they are 3-15 μm in diameter and are suitable for both intracellular and extracellular delivery. These data demonstrate that SB239063 can be successfully loaded into PK particles and the particles can be hydrolyzed for measurement of encapsulation efficiency.

The particle size can be easily changed To demonstrate the feasibility of making larger particles, we used a reduced homogenization rate using the original protocol (above). Or the emulsion was run using a reduced PVA percentage. Next, the inventors stirred at room temperature for 4 hours to evaporate the methylene chloride, washed several times, freeze-dried the polyketals, and examined their sizes using SEM. As shown in FIG. 33, by the original protocol we obtained particles in the range <10 μm. However, by reducing the homogenization rate (60 seconds, 15,000 RPM) or by reducing the PVA percentage (from 8% to 4%), we produce particles of 20 μm and larger. I was able to. This data shows that larger particles can be produced by reducing the rate of homogenization or by reducing the percentage of aqueous solution. This indicates the feasibility of generating larger particles as an alternative approach if sufficient myocardial retention is not obtained.

Porous PCADK microparticles can be synthesized With respect to pulmonary drug delivery, porous microparticles of 20-30 microns in size have recently been of attractive interest. Because their large size still prevents them from being phagocytosed by macrophages, their low density provides their porous microparticles with aerodynamic properties to be inhaled via spinhalers . A study by Langer et al. Using PLGA showed that 20-30 micron sized particles are optimal for delivering therapeutic agents to the extracellular space of the alveolar region of the lung (Cheng et al., Formulation of function materialized PLGA-PEG nanoparticulates for in vivo targeted drug delivery. Et al., Poly (lactic acid) -poly (ethylene glycol) nano . Articles as new carriers for the delivery of plasmid DNA J Control Release (2001) 75 (1-2): 211-24).

  Accordingly, the inventors have developed an experimental protocol that produces protein loaded porous PCADK microparticles. This protocol was adapted from the procedure used to formulate porous polylactide microspheres by Hong et al. Briefly, 100 mg of PCADK polymer was dissolved in 2.55 mL of methylene chloride. Next, 0.45 mL of n-hexane was added to the polymer / methylene chloride solution and homogenized at 21,500 rpm for 30 seconds to form an “oil layer”. The water-in-oil emulsion is then added by adding 200 μl of aqueous FITC-labeled OVA solution (25 mg / ml) to the oil layer and dispersing the water layer into the oil layer via homogenization at 21,500 rpm for 90 seconds. Generated. The cloudy w / o emulsion was then poured into 20 mL of a 2% (w / v) aqueous polyvinyl alcohol solution and homogenized at 17,000 rpm for 60 seconds to produce the final water-in-oil-in-water emulsion. The w / o / w emulsion is then decanted into 100 mL of 2% PVA solution, the methylene chloride in the oil layer is evaporated, and the polymer is cured at 500 rpm at 500 rpm so that it cures around the encapsulated OVA. Stir for 25 hours at 25 ° C. The cured particles were then stirred at 500 rpm for 72 hours at 40 ° C. to remove hexane from the particles, leaving pores inside and on the surface of the particles. An SEM image of OVA-PCADK is shown in FIG. 34 demonstrating that porous particles have been synthesized. These data support our alternative plan in case of problems with the release of SB239063 in vivo.

Release kinetics can be altered by copolymer addition In this study, the effect of hydrophobicity on the hydrolysis kinetics of polyketal copolymers was investigated. Two sets of copolymers were synthesized and their hydrolysis rates were investigated. Initially, a series of polyketal copolymers composed of 1,4-cyclohexanedimethanol and 1,5-pentanediol, which contain various ratios of 1,5-pentanediol, were synthesized. The hydrophilicity of these copolymers is due to the large difference in hydrophobicity between 1,5-pentanediol and 1,4-cyclohexanedimethanol (their Log P values are 0.27 and 1.75 respectively). On the basis of their 1,5-pentanediol content. We demonstrate that 1,5-pentanediol dramatically accelerates the hydrolysis kinetics of 1,4-cyclohexanedimethanol based polyketals. For example, at pH 4.5, PCADK has a hydrolysis half-life of 20 days, whereas PK1, a copolymer containing 2% pentanediol, has a half-life of 12.5 days at pH 4.5, And PK3 which is a copolymer containing 13% mol of 1,5-pentanediol has a hydrolysis half-life of only 2 days. A second series of copolymers based on 1,4-cyclohexanedimethanol containing various hydrophilic diols was also synthesized. In summary, the data in Table 3 demonstrate that the hydrolysis kinetics of polyketals can be tuned by changing their hydrophilicity, with water diffusion into the polymer matrix governing the hydrolysis of the ketal. It is suggested that this is a rate-determining step. These copolymers of PCADK are used for our study. This is because they have “tunable” hydrolysis rates and particles that slowly hydrolyze over several weeks are optimal for heart regeneration.

An experimental protocol capable of producing polyketals loaded with proteins was developed based on the procedure used to formulate SOD into PLGA-based microparticles. Briefly, 100 μL of SOD aqueous solution (40 mg / mL) was dispersed in 1.0 mL of methylene chloride containing 125 mg of PCADK by homogenization (21,500 rpm, 30 seconds) and water-in-oil (w / o) An emulsion was produced. Next, this w / o emulsion was added dropwise to 5 mL of an 8% (w / v) aqueous solution of polyvinyl alcohol (PVA), and stirred at 6,000 rpm for 5 minutes using a homogenizer. The resulting w / o / w emulsion was then poured into 25 mL pH 7.4 buffer and the mixture was stirred for several hours to evaporate the methylene chloride. The resulting particles were isolated by centrifugation and lyophilized to produce a white solid powder. The protein encapsulation efficiency of SOD-PCADK microparticles is the U.S. V. It was 36.7% as determined by absorbance. The SEM images of SOD-PCADK microparticles shown in FIG. 35 demonstrate that they are 3-15 μm in diameter and are suitable for both intracellular and extracellular delivery. This can be beneficial in the case of SOD delivery. This is because superoxide causes both intracellular toxicity and extracellular tissue damage during inflammation. These data demonstrate the versatility of polyketals in their ability to encapsulate larger, hydrophilic proteins, in addition to small molecule inhibitors, for our alternative approach.

PCADK enhances delivery of SOD to macrophages The ability of SOD-PCADK microparticles to remove superoxide from macrophages was examined in cell culture. TIB-186 macrophages (1 × 10 ⁵ cells / well, 96 well plate) with either 0.1 mg / mL SOD-PCADK microparticles, free SOD (1.14 μg / mL SOD), or empty PCADK microparticles. Incubated for 2 hours and then stimulated with 0.2 μg / mL phorbol myristate acetate (PMA) for 30 minutes. Superoxide production from these macrophages was then measured using 20 nmol cytochrome c (absorbance at 550 nm). FIG. 36 demonstrates that free SOD alone produced very little inhibition of superoxide production, whereas SOD-PCADK microparticles produced a 60% reduction in superoxide production. Empty microparticles also reduced superoxide production by macrophages by approximately 20%. These data indicate that the enzyme protein can retain its activity when encapsulated for cellular delivery. While formulated for another scheme, these data demonstrate the feasibility of polyketal-mediated delivery of proteins to cells as an alternative method.

Polyketals are retained by macrophages in culture In general, macrophages phagocytose foreign particles via phagocytosis, but our polyketals containing SB239063 do not phagocytosis. It may be too large to receive (> 5 μm) and the primary method of release may be the uptake of small molecules following particle hydrolysis by macrophages. To determine whether polyketals are retained by macrophages, we incubated FITC loaded polyketals with RAW macrophages for 2 hours, washed 3 times with PBS, and used fluorescence microscopy. Imaged. As FIG. 37 shows, FITC loaded polyketals (and unloaded polyketals) can be found outside the cells. Furthermore, FIG. 37 shows cells (arrows) that have taken up FITC. These data demonstrate that polyketals “stick” to cultured macrophages and their contents can be released following hydrolysis for uptake.

SB239063 particles inhibit phosphorylation of p38 in TNF-α-stimulated macrophages To determine the effect of PK-p38 _i on cytokine-mediated phosphorylation of p38 and superoxide release, RAW macrophages do not contain serum Plated at confluence in medium and switched to serum free medium containing empty PK or PK-p38 _i for 2, 4 or 6 hours prior to 20 minutes of TNF-α stimulation. The protein was recovered with sample buffer and 20 μg was run on a 12% SDS-polyacrylamide gel for Western analysis. The protein was transferred to a PVDF membrane and probed with an antibody against phospho-specific p38 (Cell Signaling). Blots were scanned and quantified using Quantity One software from Bio-Rad. As FIG. 38 demonstrates, PK-p38 _i (0.1 mg / mL or 2 μM) prevented p38 phosphorylation by TNF-α in a time-dependent manner. At various time points, there was no effect of empty PK, but treatment of macrophages with PK-p38 _i for 4 or 6 hours completely prevented p38 phosphorylation by TNF-α. These data suggested that PK-p38 _i may prevent cytokine-mediated activation of p38 and superoxide production in macrophages.

SB239063 particles inhibit superoxide production in TNF-α stimulated macrophages Increased superoxide production after myocardial infarction may play a role in post-infarction cardiac apoptosis and dysfunction. TNF-α stimulation can result in extracellular superoxide production in macrophages, and this response has been shown to be p38 dependent.

To determine whether polyketals loaded with SB239063 can inhibit extracellular superoxide production, we used high performance liquid chromatography to oxidize dihydroxidized in the medium of stimulated cells. adopted the published methods for measuring the accumulation of ethidium (Fernandes et al., Analysis of dihydroethidium-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems. Am J Physiol Cell Physiol (2006) , Fink et al., Detection of intr . Cellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay Am J Physiol Cell Physiol, (2004) 287 (4): C895-902). The important thing to note is that once dihydroethidium is oxidized, it can no longer cross the cell membrane, so any oxy-ethidium formed in the extracellular space remains there for measurement. Is to do. Oxy-ethidium is stable and the value can be quantified using the extinction coefficient, and the mole of superoxide is assumed to be the same as the mole of oxy-ethidium. Because they react at a 1: 1 ratio. Macrophages were plated at 10 ⁶ cells / well in serum free media the night before the experiment. The cells were then switched to serum free medium containing buffer, empty PK or PK-p38 _i for 6 hours and washed thoroughly prior to stimulation with TNF-α. TNF-α (10 ng / mL) was added to the cells in Krebs-Hepes buffer for 15 minutes, followed by dihydroethidium (25 μM) for 20 minutes. 100 μL of medium was collected in 300 μL of methanol for HPLC equipped with a fluorescence detector. As the data in FIG. 39 (n = 4) demonstrate, there is a significant increase in extracellular superoxide production in macrophages stimulated with TNF-α, which is due to pretreatment with PK-p38 _i . Although completely inhibited, it was not affected by the empty polyketal. Exogenously added superoxide dismutase (50 U / mL) completely abolished this signal. Given that extracellular superoxide release is significantly increased in response to myocardial infarction (FIG. 40), this may present a potential mechanism to be investigated in future studies. However, these data demonstrate that polyketals can deliver SB239063 to cells and not only inhibit TNF-α-induced p38 phosphorylation, but also downstream results as well.

Polyketals are retained in muscle for 3 days after injection To determine whether polyketals can be detected in vivo after several days, we have added 25 mg to the limb muscles of C57BL / 6J mice. / ML of empty polyketal or poly (lactic-co-glycolic acid) (PLGA) was injected. Three days after injection, muscles were isolated, fixed in 4% paraformaldehyde and embedded for histology. Hematoxylin & eosin staining was performed on 5 μm sections and images were acquired using light microscopy. As the representative image in FIG. 41 demonstrates, particles were detectable 3 days after injection. Interestingly, despite the unusually high concentration of particles (25 mg / mL is 25 to 250 times higher than the experiments proposed by the inventors), there is very little inflammation around the particles. It seemed. In contrast, limb muscles injected with PLGA particles showed the potential for severe fibrosis and inflammatory cell infiltration, as observed by the following figure. Note, the dark staining with the PCADK sample is mostly particles and not cells, as confirmed by DAPI staining. These data demonstrate important proof of principle in that PCADK particle injections can be confirmed by light microscopy and do not appear to result in a severe inflammatory response.

Polyketal injection does not elicit an inflammatory response To determine the effect of polyketal injection on inflammation, sections from the above limb injection experiments were stained with a fluorescent antibody against CD45 (a common marker of inflammatory cells). Specifically, after paraffin removal and rehydration, the sections were incubated with proteinase K for antigen retrieval. After recovery, sections were blocked with 10% horse serum and then incubated with fluorescently tagged anti-mouse CD45 antibody (eBioscience). After extensive washing, the sections were incubated with DAPI (1 μg / mL) and covered slips. Appropriate FITC-conjugated isotype was used as a control and was apparently free of staining. Images were acquired with exactly the same exposure time and combined using Matlab software. As the image in FIG. 42 demonstrates, there was a robust staining throughout the injection area of the PLGA treated muscle compared to the left polyketal injected muscle. We confirmed this data using staining for CD14 (another marker of inflammatory cells). Although there was a vigorous cellular response in PLGA-treated muscle, we believe that the majority of DAPI staining in PCADK-treated muscle is nonspecific. These data are essential to this proposal, demonstrating that the inflammatory response to injection of high concentrations of polyketals is negligible. Despite the possibility that the concentration used in this experiment was several orders of magnitude higher than the injection proposed by the inventors, the response of inflammatory cells as measured by CD45 and CD14 staining was negligible. These data provide the belief that any reduction we have observed in inflammation in relation to PK-p38 _i is not due to offsetting any inflammation caused by the particles themselves.

SB239063 in the polyketal inhibits phosphorylation of p38 in the infarct zone To determine if the polyketal that encapsulates SB239063 is active in vivo, C57BL / 6 mice undergoing coronary artery ligation and immediately following infarction In addition, either an empty polyketal (PK) or a polyketal containing SB239063 (PK-p38 _i 0.1 mg / mL) was given into the myocardium in a double-blind manner. The heart was excised 3 days after the infarction and the left ventricular free wall infarct zone was isolated and homogenized with Triton-X buffer containing protease and phosphatase inhibitors. 500 μg of protein was incubated overnight with polyclonal p38 antibody (Cell Signaling) and then with protein-A agarose. The beads were washed and incubated with sample buffer before running on a 12% gel. The protein was transferred to a PVDF membrane and probed with a phospho-specific p38 antibody (Cell Signaling). After development, the membrane was stripped and re-probed with total p38 antibody. Blots were scanned and quantified using Quantity One software, data expressed as the ratio of phospho-p38 to total p38, and statistically compared using ANOVA. As the blot and data in FIG. 43 demonstrated, there was a significant increase in left ventricular phospho-p38 3 days after infarction. This increase was not inhibited by empty PK but was completely abolished by PK-p38 _i treatment. These data demonstrate that SB239063 encapsulated in polyketals is active in vivo and significantly reduces phosphorylation of p38 in the infarct zone after acute myocardial infarction.

SB239063 in the polyketal inhibits the increase in IL-5 after myocardial infarction Protein homogenate (50 μg) from the above double-blind experiment was run on a 12% gel and applied to a PVDF membrane for western analysis. Moved. Interleukin-5 (IL-5; AbCam) (a pro-inflammatory cytokine associated with post-infarction cardiac dysfunction (Wei et al. The rat J Cardiovas Pharmacol, 2002. 39 (6): 842-50), and then the membrane was stripped and re-reprobed with an antibody against β-actin for normalization. 3 days after infarction as shown in representative blots and grouped data in Later, there was a significant increase in IL-5 expression, which was not disturbed by empty PK treatment, but was completely abolished by PK-p38 _i treatment These data indicate that SB239063 within the polyketal Demonstrates the potential to inhibit pro-inflammatory cytokine production after acute myocardial infarction.

SB239063 in a polyketal inhibits TNF-α increase after myocardial infarction in rats Most of the above data was collected using a mouse model, but we have We began to analyze the data from the double-blind study. To measure TNF-α release, adult male Sprague-Dawley rats were subjected to sham surgery or coronary ligation surgery and 50 μL of 0.5 mg / mL empty PK, PK-p38 _i or free SB239063 (1 μM ; Corresponding to 0.5 mg / mL PK-p38 _i ). The left ventricular free wall was harvested and homogenized 3 days after ligation and protein levels were determined by Bradford assay (Bio-Rad). Samples were then analyzed for TNF-α content using a commercially available ELISA kit (eBioscience). Data were expressed as pg / mg protein after correction. As the data in FIG. 45 shows, there was a 2-fold increase in TNF-α production in the infarcted animals 3 days after ligation. Because of the small number (n = 3-4 per group), there was a tendency for inhibition by PK-p38 _i (approximately 33% reduction), although not statistically significant. There was also no apparent effect on injection of empty polyketal (PK) or comparable amounts of free SB239063. These data support our first positive findings in mice that SB239063 encapsulation in polyketals can inhibit the production of pro-inflammatory cytokines after myocardial infarction in rats. Suggest.

Results and Discussion In this example, we presented data consisting of both particle synthesis and testing, as well as some cellular and post-infarction effects. Our data show that particles encapsulating the p38 inhibitor SB239063 can be generated from a relatively simple single emulsion protocol. Furthermore, our data clearly show that the properties of the particles can be easily changed. Specifically, by changing the homogenization rate or the aqueous solution, the particle size can be increased, ensuring retention in the myocardium. Furthermore, by adding various copolymers, we can fine-tune the hydrolysis kinetics to meet the need for post-infarction healing processes. Finally, in addition to encapsulating small molecule inhibitors, we can also encapsulate large enzyme proteins when our needs demand it.

When these particles are delivered to cultured macrophages, they are large enough to be retained out of the cells, but the contents of the polyketal particles are clearly delivered intracellularly. This occurs in a time dependent manner. This is because incubation of macrophages with PK-p38 _i took at least 4 hours to significantly inhibit phosphorylation of p38 in response to TNF-α stimulation. This inhibition was transferred to downstream effects. This is because PK-p38 _i pretreatment inhibited TNF-α-induced extracellular superoxide production.

When delivered to the limb muscles, the particles were easily distinguishable from the surrounding tissue and were retained for several days after injection. Compared to PLGA particles that degrade into acidic products, PCADK caused very little inflammation as measured by CD45 and CD14 staining. When delivered intramyocardially, PK-p38 _i reduced left ventricular p38 phosphorylation in response to myocardial infarction. Moreover, PK-p38 _i treatment reduced the level of the left ventricle of the IL-5 and TNF-alpha (2 single proinflammatory cytokines). In contrast, empty PK, or even free SB239063, had no effect.

  In summary, this example shows that retention of compound SB239063 (p38 inhibitor) in the area of infarct after a single injection inhibits cell death of endogenous cardiomyocytes in larger rodents. And restores cardiac function after myocardial infarction and improves cell therapy.

  The data herein demonstrated small micron scale polyketal (PK) particles that encapsulate SB239063 and our ability to produce TNF-α-induced p38 activation and superoxide formation in cultured cells. . Our data also showed that these particles can be delivered to the post-infarction myocardium and reduce phosphorylation of p38 in the infarct region of mice.

Example 7B: Future Studies A polyketal (PK) containing the p38 inhibitor SB239063 can inhibit p38 phosphorylation, pro-inflammatory cytokine production and apoptosis in cultured cells.

  SB239063 encapsulated in PK can be used to inhibit TNF-α-induced p38 phosphorylation and apoptosis in rat neonatal cardiomyocytes in a dose-dependent manner over a period of several days.

Experimental design: PK-p38 _i is generated as described in the method section above. We isolated rat neonatal cardiomyocytes as described in the method above and with them 0 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL empty PK or PK-p38 _i is incubated for 6 hours based on the time course shown in FIG. Following PK-p38 _i incubation, TNF-α (10 ng / mL) is added to the cells for 30 minutes and the protein is recovered in SDS-sample buffer. This dose of TNF-α should be sufficient for p38 activation in most cell types. Samples are run on an SDS-PAGE gel and probed with an antibody that recognizes either phosphorylated p38 protein or total p38 protein. This experiment is repeated with the addition of TNF-α performed 24 hours, 48 hours and 72 hours after PK-p38 _i treatment and washing. Free SB293063 is used as a positive control for inhibition. Densitometry is performed and groups are statistically compared by ANOVA. Each experiment is repeated at least 3 times for statistics.

To determine the effect on cardiomyocyte apoptosis, rat neonatal cardiomyocytes were treated with 0 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL empty PK or PK-p38 _{i for} 6 hours. Incubate. Following PK-p38 _i incubation, TNF-α (10 ng / mL) is added to the cells for 18 hours and the protein is recovered in SDS-sample buffer. We examine apoptosis by Western analysis using antibodies to cleaved caspase-3 and cleaved PARP (two well-known apoptotic pathway signaling molecules). Groups are quantified using densitometry and compared using ANOVA. In addition to the above experiments, the cells are also harvested with trypsin and annexin V staining is performed followed by flow cytometry. Data is collected and presented as the percentage of cells undergoing apoptosis and compared for statistical significance using ANOVA and Graphpad Prism software. These experiments were repeated with the addition of TNF-α performed 24 hours, 48 hours and 72 hours after PK-p38 _i treatment, and empty particles (negative control) and unencapsulated form of SB239063 (positive control). ) As a control. In addition to measuring pro-apoptosis markers, we also examine TNF-α stimulated myocytes for apoptosis using TUNEL staining followed by fluorescence imaging.

  SB239063 encapsulated in PK may inhibit phosphorylation of p38 and production of inflammatory cytokines in both RAW and isolated primary macrophages.

Experimental design: Generate PK-p38 _i as described in the methods section. We followed a 6 hour incubation with 0 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL empty PK or PK-p38 _i followed by TNF-α ( These experiments are performed using cultured RAW cells stimulated with 10 ng / mL). In one set of experiments, total protein is extracted after 30 minutes of stimulation and Western analysis is performed on phosphorylated p38 and total p38. In addition, effectors downstream of p38, such as the transcription factor NFκB, can be examined for activation. To measure cytokine production, we stimulate cells with LPS and TNF-α following a 6 hour PK incubation. Media is isolated and cytokine production is measured using the Bio-Plex cytokine assay from Bio-Rad. Selected cytokines to be measured include TNF-α, several members of the interleukin family, interferon-γ, and several other well-known inflammatory cytokines. In particular, IL-1, IL-5, IL-6 and IL-8 are involved in cardiac dysfunction after myocardial infarction. Thus, the Bio-Plex assay described above can be performed following Western analysis for confirmation. In addition to measuring the production of pro-inflammatory cytokines, we isolated mRNA from cultured and isolated cells, and we isolated the 18S or housekeeping gene (β- Real-time PCR is performed using any of (tubulin).

We predict that PK-p38 _i inhibits TNF-α-induced phosphorylation of p38 in a dose-dependent manner. Furthermore, we predict that this inhibition is evident before addition of TNF-α whether PK-p38 _i is incubated for 6 hours, 24 hours, 48 hours or 72 hours. To do. In comparison, empty PK should have no effect on p38 phosphorylation and free SB293063 should be an appropriate positive control. Encapsulation may make some inhibitors less active. In this case, we can either increase the starting amount of SB293063 or select a surrogate inhibitor. We also predict that PK-p38 _i treatment reduces apoptosis induced by TNF-α. Many studies have shown a direct link between this pathway and cleaved caspase-3 activation followed by additional apoptotic signals. We predict that PK-p38 _i reduces caspase-3 and PARP cleavage in a dose-dependent manner and prevents annexin V exposure. There are also several other methods of detecting apoptosis that can be examined if any difficulties arise, including TUNEL, DNA fragmentation and cytochrome c release. Despite the extensive literature systematic history, p38 inhibition may not be sufficient to prevent apoptosis induced by TNF-α. In this case, we can investigate the possibility of adding other inhibitors to the encapsulation procedure, such as inhibitors of c-jun-N-terminal kinase (JNK) and stress activated protein kinase (SAPK) pathways. . This does not preclude our in vivo studies. Because TNF-α is not the only cytokine produced after infarction, early studies from other laboratories suggest a powerful role for p38 in vivo.

In addition to the effects on cardiomyocytes, we have determined that PK-p38 _i treatment dose-dependently regulates p38 phosphorylation and inflammatory cytokine production in cultured and isolated macrophages stimulated with TNF-α Predictive inhibition.

  For all Western studies, the developed film is scanned and quantified using Quantity One software from Bio-Rad. All experiments are repeated at least 3 separate times and the means are compared using ANOVA followed by an appropriate post-test. Cytokine production is expressed as pg / mL and data is compared across all groups using ANOVA followed by an appropriate post-test.

A PK containing SB239063, a p38 inhibitor, can inhibit p38 phosphorylation, apoptosis of cardiomyocytes, can prevent the expression of inflammatory markers, and can improve cardiac function after myocardial infarction. SB239063 can inhibit phosphorylation and apoptosis of p38 in the infarct zone and rescue cardiac function for up to 7 days after myocardial infarction in rats. Experimental design: PK-p38 _i is described in the method section above. Generate as follows. To male Sprague-Dawley rats Adult, sham-operated or coronary artery ligation as described in the above-described method, empty PK, randomized and injected in a blinded manner by administration of PK-p38 _i or free SB293063 To do. Three and seven days after surgery, the animals were subjected to echocardiography and MRI by a trained technician under light anesthesia, and the hearts were protein studies (n ≧ 5) or immunohistologic studies at the indicated time points. Recover for any of (n ≧ 5). For protein studies, the left ventricular free wall is excised, homogenized with detergent buffer, and the protein is run on an SDS-PAGE gel and probed with an antibody that recognizes both phospho and total levels of p38 . This method has been used by our laboratory and others to reliably detect phospho-proteins in vivo after infarction (Hsieh et al., Controlled delivery of PDGF-BB for myocardial protection using injectable self- assembling peptide nanofibers.J Clin Invest (2006) 116 (1): 237-48). Protein samples are also examined for cleaved caspase-3 levels to determine the level of apoptosis. In addition to phospho-p38 measurements, blots are also inflamed like TNF-α and several members of the interleukin family, including IL-1, IL-5, IL-6 and IL-8. Probe with an antibody against the pro-inflammatory cytokine. Densitometry is performed using Quantity One software from Bio-Rad and the data are compared statistically using ANOVA followed by an appropriate post-test. The number of our animals is justified in subsequent vertebrate sections, but power analysis using the variability published by us has determined these numbers. .

  Immunohistologically, sections are probed with antibodies that recognize both phospho-p38 and a reliable cardiomyocyte marker (troponin I, α-sarcomeric actinin). We have published several papers using our standard protocol for immunohistochemistry. Briefly, tissues are fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin for sectioning. A standard microtome is used to make 5 μm sections for all studies. For staining, following rehydration, antigen retrieval is performed using proteinase K (20 μg / mL). After washing, the sections are blocked with 10% serum prior to incubation with the indicated primary antibody and an appropriate fluorescently tagged secondary antibody. Finally, sections are incubated with DAPI (1 μg / mL) for nuclear visualization. Both the intensity of phospho-p38 staining and double positive cells are quantified using ImagePro software. The intensity per unit area and the percentage of double positive cells are compared statistically by ANOVA. To measure apoptosis, tissue sections are subjected to TUNEL staining according to the manufacturer's protocol to determine the percentage of cardiomyocytes undergoing apoptosis. Data is analyzed using ImagePro software and the percentage of apoptotic cells is counted and statistically compared using ANOVA followed by an appropriate post-test.

SB239063 encapsulated in PK can prevent the onset of long-term cardiac dysfunction and fibrosis In addition to the above experiments, we have also emptied in a randomized and double-blind manner. Adult male rats (n ≧ 10) are subjected to sham surgery or coronary artery ligation prior to injection of PK, PK-p38 _i or free SB239063. At 28 and 90 days after infarction, rats are subjected to echocardiography and MRI by a trained technician under mild anesthesia to determine if recovery is evident in the chronic phase. At the indicated time points, hearts are collected in paraformaldehyde for immunohistochemical evaluation. Fibrosis is determined by picrosirius red staining followed by staining quantification using a special program written in Matlab software. This program is designed to examine pixel intensity across several regions and reduce background in the sample. Groups are statistically compared using ANOVA followed by an appropriate post-test. In addition to fibrosis staining, immunohistochemistry is performed on general markers of cardiac fibrosis, including connective tissue growth factor and transforming growth factor.

After myocardial infarction, we predict an increase in phosphorylated p38, which is inhibited by PK-p38 _i but not by empty PK or free SB293063. We expect to observe this with both extracted proteins as well as both cardiomyocyte specific inhibition and macrophage specific inhibition using immunohistochemistry. p38 inhibition may not occur in vivo after PK-p38 _i administration. We can also investigate alternative inhibitor encapsulation or increasing SB239063 loading. In addition, we can alter the porosity and hydrolysis kinetics of the particles, if required, to improve delivery kinetics. In addition to the above, we can also consider a second injection after a few weeks as a booster. Thus, we can achieve chronic p38 inhibition by the second injection. We predict this inhibition of p38 activation after myocardial infarction resulting in a significant reduction in cardiomyocyte apoptosis. This is evident by staining for apoptotic markers including cleaved caspase-3 and TUNEL labeling. We anticipate that administration of PK-p38 _i will also reduce pro-inflammatory cytokine production in the infarct region compared to empty PK or SB239063 alone. This is evident by Western analysis for specific cytokines including TNF-α, specific members of the interleukin family and interferon-γ. Western analysis may not be sensitive enough to detect the difference between these markers in vivo. In this case we can use the Bio-Plex cytokine assay from Bio-Rad or real-time PCR for the indicated markers. It is possible that treatment with PK-p38 _i does not inhibit cytokine production after myocardial infarction. In either case, these data provide valuable information about the contribution of local post-infarction signal transduction to a more comprehensive signal in disease progression.

We anticipate that these potential and beneficial effects of PK-p38 _i outlined above will result in improved cardiac function compared to empty PK and free SB239063. This is a significant increase in fractional area in the PK-p38 _i group compared to MI alone, MI with empty PK or free SB239063, and end-diastolic ( end diastatic) should be evident by a decrease in ventricular diameter and volume. Echocardiographic measurements are made from short axis images during M mode by our technicians, and volume measurements are made from long axis measurements in a blinded fashion. In addition, cardiac MRI is performed to confirm these data. Despite the reduction of pro-inflammatory cytokine production and apoptosis, a positive effect on cardiac function may not be apparent at this early time point. For this reason, we propose to run this experiment again at a longer time (after 28 days).

  Finally, the beneficial effect on the prevention of fibrosis can be obvious. This is because several studies have shown that the p38 cascade is an important mediator of cardiac fibrosis. Thus, in the future we can examine markers of cardiac fibrosis, such as the release of pro-inflammatory cytokines by ELISA or the expression of pro-inflammatory genes by real-time PCR. Published data also demonstrated a significant increase in the proliferation of adult cardiomyocytes following infusion of p38 inhibitors (Engel et al., P38 MAP kinase inhibition of adult mammalian cardiocytoses. ): 1175-87). Although the rate of growth was very slow with the p38 inhibitor, we can investigate this by injecting BrdU prior to staining with Ki67 or sacrifice. Our laboratory and others have developed a protocol for BrdU injection to measure myocyte proliferation following injury (Hsieh et al., Controlled delivery of PDGF-BB for mycardial protection using injectable self-inflating cells). peptide nanofibers.J Clin Invest, (2006) 116 (1): 237-48).

PK containing SB239063, a p38 inhibitor, can improve cardiac stem cell survival and efficacy both in culture and in vivo PK-p38 _i can improve survival and differentiation of cultured cardiac stem cells Design: We isolate cardiac stem cells as described in the methods at the end of this section. Our preliminary isolation demonstrated high purity c-kit ⁺ cardiac stem cells, as demonstrated in this method. Cultured cardiac stem cells are plated on confluent, serum-free medium and mixed with 0 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL empty PK or PK-p38 _i Incubate for hours. Following PK incubation, cells are washed and incubated with TNF-α for 30 minutes. Protein is recovered in sample buffer and Western analysis is performed on phospho-p38 and total-p38. In addition, the cells are incubated with TNF-α for 18 hours, and the cells are stained for general markers of apoptosis, including cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Apoptosis is also measured using TUNEL and Annexin-V staining. All images are quantified using the Matlab program and expressed as% positive. Western blots are scanned and quantified using Quantity One software from Bio-Rad. All experiments are repeated at least 3 times, followed by statistical comparisons between groups using ANOVA followed by an appropriate post-test.

To determine whether improved survival can enhance differentiation, cells were treated with 10% calf serum and 10 nM dexamethasone in the presence and absence of empty PK or PK-p38 _i pretreatment. Induce differentiation in the presence. This combination of calf serum and dexamethasone has been described by Linke et al. (Linke et al., Stem cells in the dog heart self self-renewing, clonogenic, and multi-ported and regenerative infertility). ) 102 (25): 8966-71) should be sufficient to induce differentiation into cardiac stem cells. The same experiment is performed in the presence of TNF-a (10 ng / mL) to determine whether an improvement in survival confers an additional benefit on differentiation. Following treatment with calf serum and dexamethasone, the cells were permeabilized and subsequently subjected to cardiac specific markers including transcription factor Nkx2.5, cardiac myosin, α-sarcomeric actinin, connexin-43 and troponin T. Fix with 80% methanol for staining. All of these are markers of heart maturation that we have previously used to determine heart phenotypes in ancestral populations. In addition to cardiac marker specific staining, we also collect cellular RNA for the above protein measurements using real-time PCR.

PK-p38 _i may improve the survival, growth and maturation of endogenous cardiac stem cells Experimental design: PK-p38 _i is generated as described in the method above. Adult male Sprague-Dawley rats are subjected to coronary artery ligation, followed immediately by empty PK, PK-p38 _i or SB239063 alone. One day, 7 days, 14 days and 28 days after coronary artery ligation, the rats are sacrificed and the heart is fixed with 4% paraformaldehyde and embedded for immunohistologic evaluation (n ≧ 5). The presence and size of c-Kit positive cells and Sca-1 positive cells in the infarct zone are analyzed using immunofluorescence and imaging software and performed in a blinded and randomized fashion. In addition, double staining for the cardiac stem cell markers c-Kit or Sca-1 and survival factor phospho-Akt is examined using double immunofluorescence. Furthermore, TUNEL and cleaved caspase-3 staining also co-localize with c-Kit and Sca-1 to determine whether PK-p38 _i delivery improves the survival of these endogenous stem cells. To measure maturation / differentiation, we examine these progenitor cells for the expression of the cardiac markers α-sarcomere actinin, troponin T, and for the presence of sarcomere. Finally, to measure replication, we examine BrdU incorporation and Ki67 staining of c-kit and sca-1 positive stem cells. Our previous study demonstrates that a single bolus injection of BrdU 2 hours prior to sacrifice is sufficient to measure proliferating cells. For the immunohistologic evaluation described above, positive cells are counted manually and in programs written in Matlab and expressed as% positive. In addition, we have created a program in Matlab to count the size of cells (we used previously to measure the size of transplanted cells) (Davis et al., Local myocardial insulin-like growth factor 1 (IGF-1): 15 with delivery of biot animated cells. In our published work, this method is reliable and has little variability in standardized assays. Groups are compared statistically by using ANOVA comparison (using appropriate post-test). All animal numbers were justified by performing a power analysis using our previously published standard deviation for immunohistochemical staining followed by cell counts.

PK-p38 _i can improve survival, growth and maturation of transplanted cardiac stem cells Experimental Design: Isolate and culture cardiac stem cells as described in the Methods section. Prior to use, cells are infected with an adenovirus encoding hemagglutanin for tracking. Immediately after coronary artery ligation in adult male Sprague-Dawley rats (n ≧ 5 per group) in a randomized, double-blind manner, cells only, cells and empty PK, cells and free SB239063 or Cells and PK-p38 _i are injected into the infarct zone for visual inspection. In all studies, 500,000 cells are used per injection. Because we have previously been successful with this amount of injection (Davis et al., Local myocardial insulin-like growth factor 1 (IGF-1)) with the witherbirritated peptide. Proc Natl Acad Sci USA (2006) 103 (21): 8155-60). Echocardiography and cardiac MRI are performed in a double-blind manner under light anesthesia by trained technicians at 3, 14, and 28 days after infarction. In addition to cardiac function, hearts are collected on days 3 and 28, fixed with 4% paraformaldehyde, and embedded for immunohistologic evaluation (n ≧ 5 per group per time point). Sections are co-stained for hemagglutinin and cleaved caspase-3 and TUNEL for assessment of apoptosis. We have previously used hemagglutining and GFP adenovirus to track cells and can stain more than 95% of the cells (FIG. 47). Data are expressed as the percentage of transplanted stem cells positive for these markers. In addition to measuring apoptosis markers, we also stain for cardiac maturation markers, including cardiac myosin, α-sarcomeric actinin and troponin T, to determine differentiation / maturation. Finally, the sections are stained with hemagglutanin and the size is measured using a program written in Matlab. All data are evaluated for statistical significance using ANOVA followed by an appropriate post-test. We compared to empty PK, PK-p38 _i significantly inhibits cleaved caspase-3 activation in TNF-α treated cells, but free SB239063 is a suitable control. Predict that it should be. This should be evident in other markers for apoptosis, including TUNEL and Annexin-V staining. In addition to the effect on survival, we also anticipate that this protection will be transferred to increased calf serum / dexamethasone-induced differentiation as measured by marker staining of mature hearts.

In addition to the effects on cultured cells, we predict that PK-p38 _i enhances the survival of endogenous stem cells after myocardial infarction. By allowing more adult and stem cells to survive post-infarction, we believe that this also results in increased stem cell maturation as measured by staining for specific markers over time. We have improved cardiac function as measured by echocardiography and MRI when rats receiving cells transplanted with PK-p38 _i compare to cells and empty PK or free SB239063. Predict that

Careful attention is given to characterization of the cells by staining with the specific lineage markers that we have described in the above method. In addition to the magnetic bead separation we used for preliminary isolation, a fluorescence activated cell sorting method has been published for the isolation of c-kit ⁺ stem cells. The original heart stem cells are frozen and the culture is routinely checked for lineage markers to ensure the appropriate cell type.

Polyketal synthesis—Poly (1,4-cyclohexane-acetone dimethylene ketal) (PCADK) is synthesized by reacting 1,4-cyclohexanedimethanol with 2,2-dimethoxypropane. PK generated from PCADK should degrade in vivo to 1,4-cyclohexanedimethanol and acetone. PCADK is synthesized in a 25 mL two-necked flask connected to a short path distillation head. 1,4-Cyclohexanedimethanol (1.0 g, Aldrich) dissolved in 10 mL warm ethyl acetate is added to 10 mL distilled benzene maintained at 100 ° C. Next, recrystallized p-toluenesulfonic acid (5.5 mg, 0.029 mmol, Aldrich) dissolved in 550 μL of ethyl acetate is added to the benzene solution. Ethyl acetate is distilled off and distilled 2,2-dimethoxypropane (900 μL, 7.4 mmol, Aldrich) is added to initiate the reaction. An additional dose of 2,2-dimethoxypropane is added via a metering funnel to make up for the distilled off 2,2 dimethoxypropane. The reaction is stopped by adding 100 μL triethylamine and precipitated with cold hexane. The crude product is vacuum filtered, rinsed with ether and hexane, and vacuum dried to produce PCADK. The recovered polymer is analyzed by GPC and ¹ H NMR.

  Polyketal copolymers consisting of cyclohexane-dimethanol and butanediol are also synthesized using the methodology described above to manipulate the hydrophobicity and hydrolysis kinetics of polyketal microparticles. Copolymers containing 3, 5 and 10 mol% butanediol were synthesized and we found that these polymers were significantly more than PCADK at pH 7.4 due to their higher hydrophilicity. Is expected to hydrolyze and release SB239063.

  Encapsulation of SB239063-The SB239063 encapsulation experiment is performed using a single emulsion procedure using dichloromethane as the oil layer and PVA as the surfactant stabilizer. A typical procedure is described below. A 500 μL solution of SB239063 (1 mg / ml) is dispersed in the organic phase (consisting of 75 mg of PCADK dissolved in 1 mL of dichloromethane). This solution is homogenized in 4% PVA at the lowest setting for 1 minute to produce a water-in-oil (w / o) emulsion. The resulting w / o emulsion is then poured into 30 mL of 1% PVA solution and mechanically stirred for several hours until the organic solvent has evaporated. The resulting particles are isolated by centrifugation, washed thoroughly and lyophilized. The particles can be assayed for inhibitor content by A320 using an empty polyketal as a control. We generated a standard curve for the SB239063 concentration that could be applied. The resulting polyketal is also dried and examined by SEM.

  Myocardial Injection / Infarction—Adult male Sprague-Dawley rats weighing 250 grams are subjected to myocardial infarction or injection surgery as described. Briefly, animals are anesthetized (1% to 3% isoflurane) and following endotracheal intubation, the heart is exposed by rib separation. Myocardial infarction is performed by ligation of the anterior ventricular artery of the left coronary artery. For PK injection or cell therapy studies, immediately after coronary artery ligation, treatment (50-100 μL) is injected through a 30 gauge needle into the infarct zone while the heart is beating. After injection, the chest is closed and the animal recovers on a heating pad. Echocardiograms are performed as described in the experimental design above. The inventor has experience with this animal model, and we have obtained a reproducible infarct measured by echocardiography. We performed several experiments in rats and FIG. 48 shows a graph of grouped data from 10 animals that underwent sham surgery or coronary artery ligation surgery. The Y-axis is the% inner diameter shortening after 3 days (a common measure of cardiac function using echocardiography).

  Neonatal cardiomyocyte isolation—By following this protocol, we were able to recover neonatal cardiomyocytes with a purity of 95-99% (Davis et al., Local myocardial insulin-like growth factor 1 . (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction Proc Natl Acad Sci U S A (2006) 103 (21): 8155-60; Davis et al, Injectable self-assembling peptide nanofibers create intramyocardial m (4): 442-50; Narmoneva et al., Endocelial cells myc ral citral sig nal sigma (4): 442-50; One day old pups of Sprague-Dawley rats (Charles River Laboratories) were sacrificed, the heart was excised, washed in Hank's buffered salt solution, and minced for 6 hours at 4 ° C. for trypsin (1 mg / mL) Put in. The resulting pellet is then dissolved in collagenase type 2 (0.8 mg / mL) for 1 hour at 37 ° C. After light centrifugation, the resulting pellet is resuspended in medium 199 containing 20% fetal calf serum and plated for 2 hours. Non-adherent cells are plated or collected on fibronectin-coated dishes for experiments and analyzed by flow cytometry using antibodies against α-sarcomeric actin to determine myocyte composition To do.

Heart Stem Cell Isolation—Adult Sprague-Dawley rat hearts are isolated, minced and incubated with collagenase type 2 (0.8 mg / ml) at 37 ° C. for 1 hour. The supernatant is isolated by centrifugation and then incubated with c-Kit antibody conjugated to magnetic beads (Dynal). After the cells reach confluence, a second selection step is performed using an antibody against Sca-1. Cell phenotype is determined using flow cytometry for c-Kit, Sca-1 and lineage markers. Lineage markers used are CD34, CD45, CD31, connexin 43, cardiac myosin and von Willebrand factor. Using this process, several researchers were able to maintain a lineage of cardiac stem cells that can be continuously regenerated and maintained in culture (Beltrami et al., Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell (2003) 114 (6): 763-76). In addition, as FIG. 49 demonstrates, our preliminary isolation procedure has begun and is extremely effective. We were able to maintain a line of c-kit ⁺ stem cells that did not stain for other lineage markers including CD45. Further work is underway to determine the absence of other markers and sensitivity to pro-death stimuli.

  Western analysis and real-time PCR--Western analysis on proteins extracted from cultured cells and infarcted areas is performed as previously described (Hsieh et al., Controlled delivery of PDGF-BB for myocardial protection using infectious selef assemblable assemblable assembly-separable assembly-semplable assembly-semplable assembly-sep-semplifying assembly-separable-self-injectable assembly-separating-self-assembling assembly-separable-sampling-injectable-separating-self-assembling-separating-separating-separating-the-slept J Clin Invest, (2006) 116 (1): 237-48). Antibodies against CTGF, TGF-β, type 1 collagen and MMP-2 are commercially available. RNA is extracted from the infarct zone using Tri-Reagent according to the manufacturer's protocol. Western analysis is performed from cell or tissue homogenates. Cells are harvested with Tris / EDTA buffer containing 0.1% Triton-X, protease inhibitor and phosphatase inhibitor. Tissue homogenates are made using similar buffers (unless a specific protocol requires a different buffer) and homogenized using a PowerGen homogenizer (Fisher) equipped with a saw-tooth generator. To do.

  Immunostaining-Hearts are isolated at indicated time points and fixed in 4% paraformaldehyde for 6 hours at room temperature. After dehydration, the heart is embedded in paraffin and 5 μm sections are made for immunohistochemistry. Paraffin is removed by immersion in xylene and the sections are probed with antibodies as described in the method above. Antibodies against cardiac markers (troponin T, α-sarcomere-actin, cardiac myosin) are commercially available and were used in our laboratory.

  Echocardiography / MRI—Echocardiography and MRI are performed under light anesthesia by an experienced technician. All measurements are taken and analyzed in a double blind fashion. The left ventricular end systolic and diastole measurements are obtained from the M-mode short axis image and confirmed with the long axis image.

(Example 8: Synthesis of polyketal copolymer and formation of particles)
In this example, the hydrolysis kinetics of poly (cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK) can be determined through its copolymerization with other diols having various hydrophilicities. Present a strategy for manipulating by controlling. Six polyketal copolymers PK1 to PK6 based on PCADK were synthesized.

Materials and Methods Polyketal copolymers were synthesized in a 25 mL two-necked flask connected to a short path distillation head. 1,4-cyclohexanedimethanol (1.04 g, 7.25 mmol), which is a diol, and any of 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, or 1,8-octanediol The heel was dissolved in 20 mL of distilled benzene and kept at 100 ° C. Recrystallized p-toluenesulfonic acid (5.5 mg, 0.029 mmol) dissolved in 550 μL of ethyl acetate was added to the benzene solution. Ethyl acetate was distilled off and distilled 2,2-dimethoxypropane (equal to the combined molar amount of the two diols) was added to initiate the reaction. Next, additional doses of 2,2-dimethoxypropane (500 μL) and benzene (2 mL) were added to the reaction via a metering funnel every hour over a 6 hour period, and 2,2-dimethoxy distilled off. Propane and benzene were offset. After 24 hours, the reaction was stopped by adding 100 μL of triethylamine. The polymer was isolated by precipitation into cold hexane and analyzed by ¹ H-NMR and GPC. In general, the resulting polymer had a number average molecular weight between 2000 and 4000 Da. Table 1 lists the composition and molecular weight of the synthesized polyketal copolymers. ¹ H NMR spectra were acquired using a Varian Mercury VX 400 MHz NMR spectrometer (Palo Alto, Calif.) Using CDCl ₃ as a solvent. The H-NMR spectra of these polyketal copolymers are summarized below as representative examples, and ¹ H-NMR of PK3 is shown in FIG. The molar ratio of 1,5-pentanediol to 1,4-cyclohexanedimethanol was obtained by taking the ratio of the respective areas under peaks a and b.

The molecular weight of these polyketal copolymers was determined by gel permeation chromatography (GPC) using a Shimadzu system (Kyoto, Japan) equipped with a UV detector. THF was used as the mobile phase at a flow rate of 1 mL / min. Polystyrene standards from Polymer Laboratories (Amherst, Mass.) (Peaks Mw = 1060, 2970 and 10680) were used to establish molecular weight calibration curves.

Hydrolysis of these polyketal copolymers was measured at 37 ° C. in water buffered at pH values of 1.0 (0.1 M HCL), 4.5 (100 mM AcOH) and 7.4 (100 mM). Briefly, 20 mg of polymer sample was placed in a 5 mL vial and 1 mL of buffer was added to each vial and mixed with a magnetic stir bar. At specific time points, 1 mL of CDCl ₃ is added to each vial, shaken vigorously, the CDCl ₃ phase is isolated and analyzed by ¹ H NMR to determine the% hydrolysis of the ketal bond in the polyketal copolymer. did.

Results and Discussion Six polyketal copolymers PK1-PK6 (Table 1, below) based on PCADK were synthesized using an acetal exchange reaction. A copolymer of PCADK was synthesized by copolymerizing 1,4-cyclohexanedimethanol and any of butanediol, pentanediol, hexanediol, and octanediol. The hydrophilicity of these diols differs from the hydrophilicity of 1,4-cyclohexanedimethanol (logP = 1.46) as evidenced by their respective logP values (Table 2, below). FIG. 51 shows the synthetic scheme of the acetal exchange reaction used to make the polyketals described above, and the degradation products generated from their hydrolysis. The synthesis of all these polyketal copolymers was achieved in one step, multi-gram scale, 50% -60% yield. In general, the introduction of diols rather than CDM did not create any complications in the synthesis, and the procedures developed for the synthesis of PCADK were suitable for the synthesis of copolymers. Importantly, all synthesized copolyketals are crystalline and thus have the potential for formulation into microparticles.

The hydrolysis kinetics of PK1-PK6 were measured at pH values of 4.5 and 7.4 to determine their behavior in the acidic environment of phagosomes and in blood. FIG. 52 demonstrates that all polyketal copolymers undergo acid-catalyzed hydrolysis, and their hydrolysis kinetics scale inversely with their hydrophobicity. PK1, PK2 and PK3 were copolymers synthesized from 1,4-cyclohexanedimethanol and 1,5-pentanediol. Their hydrophilicity is due to the large difference in hydrophobicity between 1,5-pentanediol and 1,4-cyclohexanedimethanol (whose LogP values are 0.27 and 1.46, respectively) And is proportional to their 1,5-pentanediol content. FIG. 52A demonstrates that 1,5-pentanediol dramatically accelerates the hydrolysis kinetics of 1,4-cyclohexanedimethanol based polyketals at pH 4.5. For example, only 30% of PK1 (a copolymer containing 2% pentanediol) was hydrolyzed after 10 days at pH 4.5. On the other hand, at the same pH conditions, PK2 (containing 7.5% pentanediol) is 75% hydrolyzed after 7 days, and PK3 (containing 13% pentanediol) is 50% hydrolyzed after 2 days. And completely hydrolyzed after 5 days. For all three polyketals, less than 15% of these polymers were hydrolyzed at pH 7.4 within the duration of the experiment (FIG. 52B).

In order to determine whether this is a more general phenomenon that extends beyond the copolymer of pentanediol, the polyketal copolymers PK4, PK5 and PK6 also have a different hydrophobicity from pentanediol. They were synthesized from butanediol, hexanediol and octanediol, and their hydrolysis kinetics were investigated. Table 2 demonstrates that this set of copolymers also has an inverse relationship between hydrophobicity and hydrolysis kinetics. For example, PK4 (a copolymer synthesized using 1,4-cyclohexanedimethanol and 1,4-butanediol) has faster hydrolysis kinetics among all the copolyketals synthesized. And has a hydrolysis half-life of 1 day at pH 4.5, which is expected based on the high hydrophilicity of butanediol compared to other diols. On the other hand, PK6 synthesized using 1,4-cyclohexanedimethanol and the more hydrophobic monomer 1,8-octanediol has a hydrolysis half-life at pH 4.5 of 18.6 days. Had. Taken together, these data demonstrate that the hydrolysis kinetics of these polyketals can be adjusted by changing their hydrophobicity, and the diffusion of water into these polyketals dominates the hydrolysis Suggests that it is the rate-limiting step. Importantly, the hydrolysis kinetics of all polyketal copolymers are pH sensitive and generally they hydrolyzed at least an order of magnitude faster than pH 7.4 at pH 4.5.

  One of the above synthesized polyketal copolymers composed of cyclohexane-dimethanol and pentanediol (referred to as PK3 in Table 1) has a hydrolysis half-life of 2 days at pH 4.5, but pH 7. 4 has a hydrolysis half-life of several weeks. The polyketal can be useful for delivering a therapeutic agent to a subject. Because it should hydrolyze rapidly after phagocytosis, it is relatively stable at physiological pH. Furthermore, PK3 may be suitable for microparticle drug delivery because of its rapid hydrolysis kinetics and biocompatible degradation products that are 1,5-pentanediol, 1,4-cyclohexanedimethanol and acetone .

  For further studies using PK3 for drug therapy delivery, microparticles were formulated from PK3 using a solvent evaporation procedure. FIG. 53 demonstrates that microparticles can be formed with PK3, which particles have a size between 1 and 5 microns that is suitable for phagocytosis by macrophages. Thus, the therapeutic agent can be encapsulated in PK3 microparticles (eg, by using a water / oil / water double emulsion procedure or other methods) for administration to a subject.

  Active particles loaded microparticles are formulated from polyketal copolymers using a modified water / oil / water emulsion method (Ando et al., Journal of Pharmaceutical Sciences (1999) 88, 126-130). For example, PK3 (100 mg) is dissolved in 1 mL of dichloromethane, and in a separate vial, 40 mg of active substance is added to 400 μL of D.I. I. Dissolve in water. This aqueous solution of the active substance is mixed with the PK3 solution and subjected to ultrasonic treatment (Misonix Incorporated, Farmigdale, NY) for 60 seconds. The sonicated mixture is then immersed in liquid nitrogen for 15 seconds and 12 mL of 5% w / w PVA solution (pH 7.45) is added thereto. This mixture is homogenized for 120 seconds using a Powergen 500 homogenizer (Fisher Scientific, Waltham, Mass.) And then transferred to a beaker containing 40 mL of 1% w / w PVA (pH 7.45). The solution is then stirred for 3 hours using a magnetic stir bar to evaporate the organic solvent. The particles are isolated by centrifugation at 10,000 rpm for 15 minutes, washed twice with 15 mL PBS buffer and lyophilized.

  Reference for Example 8

Example 9: Formation of particles with a linker In this example, the polyketal molecules of the present invention were modified using a linker capable of binding an active agent. Specifically, a nitrilotriacetic acid (NTA) linker was added to PCADK. When loaded with nickel (Ni), the NTA linker allowed binding of histidine-tagged proteins (FIG. 54). The modified PCADK then formed microspheres ideal for rapid delivery of active agents (eg, growth factors) (FIG. 55).

Materials and Methods Synthesis of PCADK Particles Containing NTA Linkers PCADK has been previously synthesized as described above (Lee S et al., Polymicroparticulates: a new delivery vehicle for superjumjet. 18 (1): 4-7, and Heffman MJ and Murthy N. Polyketal nanoparticulates: a new pH-sensitive biodegradable drug delivery vehicle (N). Which are incorporated herein by reference) See also).

  After synthesis of the polyketal, 90 mg of PCADK was dissolved in 1 mL of a 10 mg / mL dichloromethane solution (final concentration of 10% DOGS-NTA) of DOGS-NTA (Avanti Polar Lipids). To ensure solubility, the mixture was sonicated for several seconds / minutes in a water bath sonicator. The polymer solution was then poured into a vial containing 5 mL PVA (2%) and homogenized for 60 seconds. The resulting solution was stirred with 40 mL of 0.5% PVA for 4-6 hours. The particles were then centrifuged, washed several times with deionized water and frozen in liquid nitrogen for lyophilization. The resulting lyophilized particles were imaged using SEM and shown in FIG.

Nickel loading of PCADK-NTA In order to obtain particles that could bind to His-tagged proteins, it was necessary to load the particles with nickel. Therefore, the particles were incubated with 10 mg / mL 50 mM NiCl _{2 and} stirred for 2 hours at room temperature. To determine nickel uptake, we incubated the particles with NiCl ₂ overnight, centrifuged the particles and measured unbound nickel in the supernatant spectrophotometrically. As the data in FIG. 57 demonstrates, at various NTA loadings, the free nickel in the solution was reduced, indicating that the particles were loaded with nickel.

Binding of His-tagged proteins to PCADK-NiNTA To determine whether NiNTA on particles can bind His-tagged proteins, various concentrations of His-GFP were applied overnight at 4 ° C. Incubated with PCADK-NiNTA (1% solution w / v). The particles were centrifuged, washed several times with PBS and then resuspended for analysis. FIG. 58 is a representative fluorescent stain for GFP converted to grayscale for display. As the image shows, these particles retained GFP on their surface. To further quantify the amount of bound His-GFP, the particles are loaded with increasing amounts of His-GFP, washed several times and an ELISA is performed on these particles to determine the level of binding. did. As shown by the data in FIG. 59 (1% NTA) and the data in FIG. 60 (10% NTA), PCADK-NiNTA bound His-tagged protein in a dose-dependent manner. In 1% NTA, these particles were saturated in binding with 60 ng His-GFP loading, while in 10% NTA, saturation was reached with 120 ng His-GFP loading. At most points in the linear range, the binding was approximately 50% effective. The data in FIG. 62 shows that the percent binding of His-GFP increases with the amount of NTA. FIG. 28 shows that His-GFP binding to NTA is stable for at least 15 hours.

  To determine if binding was reversible, particles loaded with His-GFP at various NTA concentrations were incubated with imidazole (200 mM), a competitor of the Ni-NTA complex. Supernatants were collected at various time points and assayed for GFP fluorescence. As the data in FIG. 61 demonstrate, His-GFP was released from the particles rather quickly and most of the protein was released in 30 minutes. This suggests that the protein should not have irreversible binding to the particles but should have good bioavailability.

Claims (44)

  A polyketal polymer which is PK1, PK2, PK3, PK4, PK5 or PK6.   A biodegradable particle comprising the polyketal polymer according to claim 1.   The particle of claim 2 further comprising one or more active agents. (A) one or more PK1, PK2, PK3, PK4, PK5 or PK6, and (b) an active substance,
Containing biodegradable particles.   The particle according to claim 2 or 4, which is a nanoparticle or a microparticle.   5. A particle according to claim 2 or 4 having a size of about 50 nm to 1000 μm or a size of about 200 nm to 600 μm.   The particle according to claim 3 or 4, wherein the active substance is a therapeutic agent, a preventive agent or a diagnostic agent.   The particle according to claim 7, wherein the therapeutic agent is an immunomodulator. 9. The particle of claim 8, wherein the immunomodulator is
a. A ligand for any of TLR2, 3, 4, 5, 7, 8, 9, 10 and 11, or a combination thereof,
And b. Inhibitors of regulatory pathways in dendritic cells, macrophages or antigen presenting cells c. Particles selected from the group consisting of any C-type lectin, including RIG-1, dectin-1 and DC-SIGN, or a ligand for caterpillar protein.   10. The particle of claim 9, wherein the inhibitor comprises (a) ERK, c-Fos, Foxp3, PI3 kinase, Akt, JNK, p38, NF-Kb, STAT 1, STAT2, IRF3, IRF7, IFN- an inhibitor of α signaling; or (b) a particle selected from the group consisting of inhibitors of SOCS 1, 2, 3, or other SOCS proteins.   The particle according to claim 3 or 4, wherein the active substance is a protein, peptide, carbohydrate, nucleic acid, small molecule, imaging agent, vaccine antigen and / or vaccine.   12. The particle according to claim 11, wherein the protein is a growth factor, cytokine, antioxidant protein, antibody, erythropoietin (EPO), receptor ligand.   The particle according to claim 11, wherein the nucleic acid is DNA, RNA, antisense oligonucleotide, or siRNA.   12. The particle of claim 11, wherein the small molecule is a kinase inhibitor, phosphatase inhibitor, receptor blocker, small molecule antioxidant mimic, cytoskeletal reconstitution inhibitor, receptor ligand.   The particle according to claim 2 or 4, further comprising a linker.   16. Particles according to claim 15, wherein the linker is streptavidin, RDG, GST, ssDNA, hemoglobin, amino acid sequence, antibody, polycarboxylic acid and / or chelating agent.   17. The particle of claim 16, wherein the chelator is a nitrilotriacetic acid (NTA) ligand, bipyrador or EDTA.   16. A particle according to claim 15, wherein the linker binds to a protein, peptide and / or carbohydrate.   19. A particle according to claim 18, wherein the protein, peptide and / or carbohydrate comprises a histidine tag.   20. The particle of claim 19, wherein the histidine tag comprises about 2-8 histidines.   20. The particle of claim 19, wherein the histidine tag comprises about 6 histidines.   19. A particle according to claim 18, wherein the protein is a therapeutic protein, a prophylactic protein or a diagnostic protein.   23. The particle of claim 22, wherein the therapeutic protein, prophylactic protein or diagnostic protein is a growth factor, cytokine, antioxidant enzyme or antibody, erythropoietin (EPO), receptor ligand.   A pharmaceutical composition comprising the polyketal polymer of claim 1.   A pharmaceutical composition comprising the particles according to claim 3 or 4.   A method for delivering an active substance to a subject comprising the step of administering to the subject a particle according to claim 3 or 4 wherein the active substance is released and the subject Wherein the method is degraded in the subject to be delivered to the subject.   27. A method of treating a subject afflicted with a disease or disorder by delivering an active substance that can affect the disease or disorder according to the method of claim 26.   28. The method of claim 27, wherein the disease or disorder is selected from the group consisting of autoimmune diseases, allergic diseases, infectious diseases, transplants, diabetes and cancer related disorders or complications.   29. The method of claim 28, wherein the infection is selected from the group consisting of HIV, malaria, TB, SARS, anthrax, Ebola, influenza, avian influenza and HCV.   30. The method of claim 28, wherein the autoimmune disease is selected from the group consisting of lupus, rheumatoid arthritis, psoriasis, asthma and COPD.   A method for delivering a protein, peptide and / or carbohydrate to a subject comprising administering to the subject the particle of claim 18, wherein the particle comprises the protein, peptide and / or carbohydrate. Is released and degraded in the subject so that it is delivered to the subject.   32. A method of treating a subject afflicted with a disease or disorder by delivering a protein, peptide and / or carbohydrate that can affect the disease or disorder according to the method of claim 31.   35. The method of claim 32, wherein the disease or disorder is selected from the group consisting of autoimmune diseases, allergic diseases, infectious diseases, transplants, diabetes and cancer related disorders or complications.   34. The method of claim 33, wherein the infectious disease is selected from the group consisting of HIV, malaria, TB, SARS, anthrax, Ebola, influenza, avian influenza and HCV.   34. The method of claim 33, wherein the autoimmune disease is selected from the group consisting of lupus, rheumatoid arthritis, psoriasis, asthma and COPD. A method for making a particle according to claim 3 or 4, wherein the method comprises:
a. Forming a PK1, PK2, PK3, PK4, PK5 and / or PK6 polymer; and
b. Forming one or more polymer particles of a) in the presence of one or more active substances;
And thereby producing the particles of claim 3 or 4.   The method according to claim 36, wherein the active substance is a therapeutic agent, a prophylactic agent or a diagnostic agent.   38. The method of claim 37, wherein the therapeutic agent is an immunomodulator. 40. The method of claim 38, wherein the immunomodulator is
a. A ligand for any of TLR2, 3, 4, 5, 7, 8, 9, 10 and 11, or a combination thereof,
b. Inhibitors of regulatory pathways in dendritic cells, macrophages or antigen presenting cells,
c. A method selected from the group consisting of ligands for any C-type lectin, such as RIG-1, dectin-1 and DC-SIGN, or any caterpillar protein.   40. The method of claim 39, wherein the inhibitor is (a) ERK, c-Fos, Foxp3, PI3 kinase, Akt, JNK, p38, NF-Kb, STAT1, STAT2, IRF3, IRF7, IFN-α. Or (b) a method selected from the group consisting of inhibitors of SOCS 1, 2, 3, or other SOCS proteins.   37. The method of claim 36, wherein the active agent is a protein, peptide, carbohydrate, nucleic acid, small molecule, imaging agent, vaccine antigen and / or vaccine.   42. The method of claim 41, wherein the protein is a growth factor, cytokine, antioxidant protein, antibody, erythropoietin (EPO) or receptor ligand.   42. The particle of claim 41, wherein the nucleic acid is DNA, RNA, antisense oligonucleotide or siRNA.   42. The particle of claim 41, wherein the small molecule is a kinase inhibitor, phosphatase inhibitor, receptor blocker, small molecule antioxidant mimic, cytoskeletal reconstitution inhibitor or receptor ligand.