WO2018027103A1

WO2018027103A1 - Compositions for microwave irradiation controlled drug release and methods of use

Info

Publication number: WO2018027103A1
Application number: PCT/US2017/045439
Authority: WO
Inventors: Guihua Yu; Ye Shi
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2016-08-04
Filing date: 2017-08-04
Publication date: 2018-02-08

Abstract

The present invention relates to compositions and methods for controlled release of an agent. In certain aspects, the invention comprises a microcarrier and at least one agent therein. The microcarrier is comprised of a shell comprising a thermo-sensitive material and a core comprising a microwave-sensitive material. In one embodiment, the microcarrier releases the agent under microwave irradiation thereby allowing allows for specific delivery of the agent when the local environment dictates.

Description

TITLE OF THE INVENTION

Compositions for Microwave Irradiation Controlled Drug Release and Methods of

Use CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Patent

Application Serial No. 62/370,897, filed August 4, 2016, the entire contents of which is incorporated herein by reference. BACKGROUND OF THE INVENTION

Smart micro-/nano-scale carrier systems for targeted drug release have been tremendously researched in the field of nanomedicine (Paul, et al, ACS Nano, 2014, 8:8050-8062; Bertrand, et al, Control. Release, 2012, 161 : 152-163; Bae, et al, J. Control. Release, 2011, 153: 198-205; Gaharwar, et al, J. Control. Release, 2014, 187:66-73). The purpose for efficient, targeted drug release is to increase the concentration of the medication in targeted parts of the body, thus avoiding the weakening of drug efficacy altered by nonspecific cell and reducing the side effects of drugs to non-targeted tissues (Mura, et al., Nat. Mater., 2013, 12:991-1003).

MicroCarriers for targeted drug delivery and release are usually constructed based on the properties of materials which could respond to either internal or external triggers (Ganta, et al, J. Control. Release, 2008, 126: 187-204; Wang, et al, Nat. Mater., 2011, 10:482-483). However, most of traditional thermal-responsive carriers can only be passively heated through heat transfer from surrounding media such as hot water.

Microwave responsive materials can potentially meet the requirements to actively respond to microwave trigger and realize smart drug carrier systems. These materials including inorganic ones (Petro, et al, Inorg. Mater., 2001, 37:93-98) and organic counterparts such as conductive polymers (Wu, et al, Polym. Eng. Sci., 1997, 37:738-743; Wu, et al, Science, 1994, 264: 1757-1759) could strongly absorb electromagnetic radiation and convert it to thermal energy (Li, et al, Nano Lett., 2015, 15: 1146-115; Shi, et al, Adv. Funct. Mater., 2015, 25: 1219-1225). Conductive polymers have been widely used for energy (Shi, et al, Chem. SOC. Rev. 2015, 44:6684-6696), electronics (Shi, et al, Nano Lett., 2015, 15: 6276-6281), and biosensor devices (Li, et al., J. Mater. Chem. B, 2015, 3:2920-2930). Microwave irradiation is a promising external trigger for smart drug release because it is noninvasive, possesses high thermal efficiency, can penetrate deep into the interior of the body, and can be accurately and remotely controlled (Wong, Curr. Drug Deliv., 2008, 5 :77-84; Qiu, et al, J. Phys. Chem. C, 2014, 1 18: 14929-14937; Wong, et al, Int. J. Pharm., 2008, 357: 154-163; Wong, et al, J. Control. Release, 2005, 104:461- 475). For a microwave triggered carrier system, both the adoption of component materials and the design of microstructures play important roles in ensuring its high efficiency.

There is thus a need in the art for sensitive and efficient thermoresponsive drug carriers, and hybrid material systems which have the ability to actively respond to external triggers. The present invention addresses this unmet need in the art.

SUMMARY OF THE INVENTION

In one aspect, this invention relates to a composition for controlled release of an agent, the composition comprising at least one microcarrier having a shell and a core, wherein the microcarrier comprises an effective amount of at least one agent.

In one embodiment, the shell comprises a thermoresponsive material. In one embodiment, the shell comprises the at least one agent. In one embodiment, the thermoresponsive material is selected from the group consisting of poly(N- isopropylacrylamide) (PNIPAM), poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA), hydroxypropylcellulose, poly(vinylcaprolactame), polyethylene oxide, polyvinylmethylether, polyhydroxyethylmethacrylate and polyvinyl methyl ether. In one embodiment, the thermoresponsive material is PNIPAM.

In one embodiment, the core comprises a microwave responsive material. In one embodiment, the microwave responsive material is selected from the group consisting of poly(p-phenylenediamine) (PpPD), polyaniline and polypyrrole.

In one embodiment, the at least one agent is selected from the group consisting of at a therapeutic agent, an imaging agent, a diagnostic agent, a contrast agent, a labeling agent, and a disinfectant agent. In one embodiment, the therapeutic agent is selected from the group consisting of a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, and an antisense nucleic acid molecule. In one aspect, the invention relates to a method for controlling the release of an agent comprising administering to a subject an effective amount of a composition comprising a microcarrier wherein the microcarrier comprises at least one agent, and administering an effective amount of microwave irradiation to the subject, wherein the microcarrier releases the at least one agent after microwave irradiation. In one embodiment, the microwave irradiation is administered to a target location, wherein the microcarrier releases the at least one agent in the target location. In one embodiment, the microwave irradiation is administered at a selected time, wherein the microcarrier releases the at least one agent at the selected time. In one embodiment, the microcarrier comprises a shell and a core.

In one embodiment, the shell comprises a thermoresponsive material. In one embodiment, the thermoresponsive material is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA), hydroxypropylcellulose, poly(vinylcaprolactame), polyethylene oxide, polyvinylmethylether, polyhydroxyethylmethacrylate, and polyvinyl methyl ether.

In one embodiment, the shell comprises the at least one agent. In one embodiment, the at least one agent is selected from the group consisting of at a therapeutic agent, an imaging agent, a diagnostic agent, a contrast agent, a labeling agent, and a disinfectant agent. In one embodiment, the at least one therapeutic agent is selected from the group consisting of a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, and an antisense nucleic acid molecule.

In one embodiment, the core comprises a microwave responsive material. In one embodiment, the microwave responsive material is selected from the group consisting of poly(p-phenylenediamine) (PpPD), polyaniline, and polypyrrole.

In one aspect, the invention relates to a method of releasing an agent into a fluid comprising administering to a fluid an effective amount of a composition comprising a microcarrier wherein the microcarrier comprises at least one agent, and administering an effective amount of microwave irradiation to the fluid, wherein the microcarrier releases the at least one agent after microwave irradiation. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and

instrumentalities of the embodiments shown in the drawings.

Figure 1 depicts an illustration of the core-shell structure of

PpPD/PNIPAM microcarriers and their drug loading and releasing processes. The drug is loaded into PNIPAM shell by a deswell-reswell process and this loading could be readily repeated. The controlled release of drug is triggered by microwave irradiation.

Figure 2, comprising Figure 2A through Figure 2D, depicts SEM and STEM images microcarriers. Figure 2A depicts SEM images of typical PpPD microparticles. Figure 2B depicts SEM images of typical PpPD/PNIPAM

microparticles. Figure 2C depicts a STEM image of PpPD particles. Figure 2D depicts a STEM image of PpPD/PNIPAM microparticles' core-shell structure. The dashed line indicates the boundary between PpPD core and porous PNIPAM shell.

Figure 3, comprising Figure 3A and Figure 3B, shows the side view SEM and STEM images of PpPD/PNIPAM microparticles. The surface of hybrid particles became rough and an outer layer with highly porous structure was formed. Figure 3 A depicts a side view SEM image of PpPD/PNIPAM microparticles. Figure 3B depicts a side view STEM image of PpPD/PNIPAM microparticles.

Figure 4, comprising Figure 4A through Figure 4D, depicts analysis of PNIPAM, PpPD, and PpPD/PNIPAM microparticles. Figure 4A depicts FTIR spectra comparing PpPD and PpPD/PNIPAM microparticles. Figure 4B depicts XRD patterns of PNIPAM, PpPD, and PpPD/PNIPAM microparticles. Figure 4C depicts UV-vis spectra of PNIPAM, PpPD, and PpPD/PNIPAM microparticles. Figure 4D depicts TGA curves of PNIPAM, PpPD, and PpPD/PNIPAM microparticles.

Figure 5 depicts the Raman spectra of PpPD and PpPD/PNIPAM microparticles. The band between 1510 cm^"1 and 1594 cm^"1 could be ascribed to the N-H bending deformation mode and the C-C deformation of benzenoid rings and quinoid rings. Figure 6 depicts the XRD pattern of PDA monomers. The spectrum showed sharp peaks at 12°, 17°, 19°, 22°, 24°, 26°, 26.5°, 27°, 29°, and 32° which were different from those of PpPD particles, confirming the successful polymerization of PpPD microparticles.

Figure 7 depicts the TGA test for PDA monomers. Before polymerization to PpPD particles, PDA monomers showed a significant weight loss in the range of 150 to 240 °C, which is much lower than those of PpPD and

PpPD/PNIPAM microparticles. The results demonstrated that the thermal stability was significantly enhanced after polymerization.

Figure 8 depicts results of a cell viability experiment using HEK293 cells treated with different concentrations of PpPD/PNIPAM microparticles by CCK- 8 assay. The cytotoxicity was examined through testing the cell viability upon exposure to the hybrid microparticles by using the standard cell counting Kit-8 (CCK- 8) assay. No apparent reduction in cell viability was found after incubation of the cells with the hydrogel blends at concentrations up to 100 μg/mL.

Figure 9, comprising Figure 9A through Figure 9D, depicts microwave heating tests for PpPD and PpPD/PNIPAM microparticles. Figure 9A depicts microwave heating at 120W test for DI water of PpPD and PpPD/PNIPAM microparticles. Figure 9B depicts microwave heating at 360W test for DI water of PpPD and PpPD/PNIPAM microparticles. Figure 9C depicts the optical images of vials without and with PpPD/PNIPAM microparticles after 10 seconds microwave irradiation. Figure 9D depicts the temperature mapping images of vials without and with PpPD/PNIPAM microparticles after 10 seconds microwave irradiation.

Figure 10, comprising Figure 10A and Figure 10B, depicts results of controlled drug release tests of PpPD/PNIPAM microcarriers. Figure 10A depicts controlled release of folic acid. Figure 10B depicts controlled release of etoposide. Efficient release of both drugs was achieved by using PpPD/PNIPAM microparticles and triggered by microwave.

Figure 1 1 depicts UV-vis spectra for samples collected during a typical controlled release test. The spectra with solid line represented the samples collected for the irradiation stages and the spectra with dash line represented the samples collected for the non-irradiation stages. The drug used here was folic acid.

Figure 12, comprising Figure 12A through Figure 12D, depicts microwave heating tests for PpPD and PpPD/PNIPAM microparticles. Figure 12A depicts microwave heating at 120W tests for PpPD and PpPD/PNIPAM

microparticles in DI and PBS-buffered water. Figure 12B depicts microwave heating at 360W test of PpPD and PpPD/PNIPAM microparticles in DI and PBS-buffered water. Figure 12C depicts the optical images of vials without and with

PpPD/PNIPAM microparticles after 10 seconds microwave irradiation. Figure 12D depicts the temperature mapping images of vials without and with PpPD/PNIPAM microparticles after 10 seconds microwave irradiation.

Figure 13, comprising Figure 13A and Figure 13B, depicts results of controlled drug release tests of PpPD/PNIPAM microcarriers in water and PBS buffer solution. Figure 13A depicts controlled release of folic acid. Figure 13B depicts controlled release of etoposide. Efficient release of both drugs was achieved by using PpPD/PNIPAM microparticles and triggered by microwave.

Figure 14 depicts reflection loss experiments of PpPD-containing films of various thicknesses at the 2-18 GHz band.

Figure 15 depicts the results of controlled drug release tests of

SiCh/PNIPAM carriers for folic acid and etoposide.

Figure 16 depicts a STEM image of SiCh/PNIPAM particles.

DETAILED DESCRIPTION

The present invention relates generally to compositions and methods for controlled delivery of an agent, such as a therapeutic agent. In one embodiment, the delivery of an agent is triggered by microwave irradiation.

In one embodiment, the invention is a composition comprising a microcarrier comprising an agent wherein the microcarrier releases the agent upon microwave irradiation. In one embodiment, the microcarrier comprises a core and a shell. In one embodiment, the core comprises a conductive or microwave-sensitive material and the shell comprises a thermo-responsive material. Examples of microwave-sensitive materials include, but are not limited to poly(p- phenylenediamine). Examples of thermo-sensitive materials include, but are not limited to poly(N-isopropylacrylamide). Accordingly, in certain instances, when the microcarrier is exposed to microwave irradiation, the microwave-sensitive material generates a local thermal field in the microcarrier. The heat given off by the microwave-sensitive material heats the thermo-responsive material and releases the therapeutic agent. Thus, the microcarrier provides targeted drug-delivery at the site of the microwave irradiation.

In some embodiments, the agent is contained within the shell of the microcarrier. In certain embodiments, the microcarrier is capable of releasing charged and uncharged agents. In some embodiments, the microcarrier comprises a therapeutic agent. For example, in one embodiment the microcarrier comprises a charged therapeutic agent, such as folic acid. In one embodiment, the microcarrier comprises an uncharged therapeutic agent, such as etoposide. In one embodiment, the microcarrier comprises a therapeutic agent such as a protein, a peptide, a

peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, or an antisense nucleic acid molecule.

The invention also provides a method for controlling the release of a therapeutic agent comprising administering to a subject an effective amount of a composition comprising microcarrier wherein the microcarrier comprises at least one therapeutic agent, and administering an effective amount of microwave irradiation to the subject, wherein the microcarrier releases the at least one therapeutic agent after microwave irradiation.

In some embodiments, the microwave irradiation is administered to a targeted location, such as a targeted tissue or targeted organ. Accordingly the microcarrier releases the at least one therapeutic agent at the targeted location while simultaneously not releasing the at least one therapeutic agent at locations outside of the targeted location which were not exposed to the microwave irradiation.

In one embodiment, the method of the invention also provides for temporal release of the at least one therapeutic agent. In one embodiment, the microwave irradiation is administered at a specific time, such that the microcarrier releases the at least one therapeutic agent at the specified time and retaining the at least one therapeutic agent prior to microwave irradiation.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, a "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

As used herein, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein with respect to the compositions of the invention, "biologically active" means that the compositions elicit a biological response in a mammal that can be monitored and characterized in comparison with an untreated mammal.

As used herein, the term "treating" means ameliorating the effects of, or delaying, halting or reversing the progress of a disease or disorder. The word encompasses reducing the severity of a symptom of a disease or disorder and/or the frequency of a symptom of a disease or disorder.

As used herein, the term "prevent" or "prevention" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease. Disease and disorder are used interchangeably herein.

As used herein, the term "medical intervention" means a set of one or more medical procedures or treatments that are required for ameliorating the effects of, delaying, halting or reversing a disease or disorder of a subject. A medical intervention may involve surgical procedures or not, depending on the disease or disorder in question. A medical intervention may be wholly or partially performed by a medical specialist, or may be wholly or partially performed by the subject himself or herself, if capable, under the supervision of a medical specialist or according to literature or protocols provided by the medical specialist.

As used herein, the terms "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" of a composition are used interchangeably to refer to the amount of the composition that is sufficient to provide a beneficial effect to the subject to which the composition is administered. The term to "treat," as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject or administering a composition to reduce the severity with which symptoms are experienced. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

By the term "specifically bind" or "specifically binds," as used herein, is meant that a first molecule (e.g., an antibody) preferentially binds to a second molecule (e.g., a particular antigenic epitope), but does not necessarily bind only to that second molecule.

As used herein, a "prophylactic" or "preventive" treatment is a treatment administered to a subj ect who does not exhibit signs of a disease or disorder or exhibits only early signs of the disease or disorder for the purpose of decreasing the risk of developing pathology associated with the disease or disorder.

As used herein, a "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology of a disease or disorder for the purpose of diminishing or eliminating those signs.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, a "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;

Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein

"pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the compositions.

As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

As used herein, the term "polymer" refers to a molecule composed of repeating structural units typically connected by covalent chemical bonds. The term "polymer" is also meant to include the terms copolymer and oligomers. In one embodiment, a polymer comprises a backbone (i.e., the chemical connectivity that defines the central chain of the polymer, including chemical linkages among the various polymerized monomeric units) and a side chain (i.e., the chemical connectivity that extends away from the backbone).

As used herein, the term "subject" refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like). In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an "individual" or a "patient." The terms "individual" and "patient" do not denote a particular age.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention is partly based upon the surprising discovery that a microcarrier, comprising a conductive core and a thermoresponsive shell and containing an agent in the shell, releases the agent upon microwave irradiation. The conductive core absorbs microwave irradiation to generate a thermal field within the microcarrier and thereby heats the thermoresponsive shell triggering the release of the agent through phase transformation of the thermoresponsive shell at high

temperatures. Thus, release of the agent from the microcarriers may be specifically targeted to specific regions by applying microwave irradiation to specific tissues. In one embodiment the conductive core will generate a thermal field within the microcarrier when microwave irradiation is applied at a frequency of about 100 MHz to about 500 GHz. In one embodiment the conductive core will generate a thermal field within the microcarrier when microwave irradiation is applied at a frequency of about 1 GHz to about 100 GHz. In one embodiment, the thermoresponsive shell will release the agent when at a temperature of about 20°C to about 1 10°C. In one embodiment, the thermoresponsive shell will release the agent when at a temperature of about 30°C to about 90°C.

The present invention provides compositions and methods for controlled drug release through microwave irradiation. In one embodiment, the composition comprises at least one microcarrier. In some embodiments, the microcarrier is comprised of a core and a shell, wherein the core comprises a microwave sensitive material and the shell comprise a thermoresponsive material. Thus, the release of contents from the microcarrier is able to be controlled.

In certain embodiments, the composition comprises at least one microcarrier and at least one agent loaded in the at least one microcarrier. In one embodiment, the microcarrier retains the at least one agent until it is triggered to release the at least one agent. This allows the at least one agent to be released only when and where it is most needed, providing increased concentration of the at least one agent in the targeted tissue or location and allowing for reduced side effects to non-targeted tissues or locations. For example, the microcarrier may be triggered to release the at least one agent by irradiation, such as microwave irradiation.

In one embodiment, the microcarrier is irradiation-activated, in which the at least one agent is released dependent on the irradiation of the local

environment. In certain embodiments, the agent is released when the microcarrier is within a local environment that is undergoing microwave irradiation. The present invention is partly based upon the inclusion of a conductive or a microwave responsive core within the microcarrier. As such, the composition of the invention provides a controllable release of an agent, by employing microwave irradiation to a localized or targeted area thereby making the agent available precisely when and where it is most needed. In one embodiment, the rate of release of the agent is dependent upon the particular composition of the microcarrier. As such, the present invention encompasses a variety of microcarrier compositions that are tailored for specific release rates at levels of microwave irradiation. In one embodiment the ratio of core material to shell material in the microcarrier is about 25: 1 to about 1 :25. In one embodiment the ratio of core material to shell material in the microcarrier is about 10: 1 to about 1 : 10.

In one embodiment, the microcarrier comprises at least one agent. In some embodiments, the agent is a therapeutic agent, an imaging agent, diagnostic agent, contrast agent, a labeling agent, a detection agent, or a disinfectant. The agent may also include substances with biological activities which are not typically considered to be active ingredients, such as fragrances, sweeteners, flavorings and flavor enhancer agents, pH adjusting agents, effervescent agents, emollients, bulking agents, soluble organic salts, permeabilizing agents, anti-oxidants, colorants or coloring agents, and the like. In one embodiment, the microcarrier comprises at least one therapeutic agent. In one embodiment, the at least one therapeutic agent is contained within the thermosensitive shell of the microcarrier. The present invention is not limited to any particular therapeutic agent, but rather encompasses any suitable therapeutic agent that can be embedded within the microcarrier. Exemplary therapeutic agents include, but are not limited to, anti-viral agents, anti-bacterial agents, chemotherapeutic agents, anti-inflammatory agents, antiseptics, anesthetics, analgesics, pharmaceutical agents, small molecules, peptides, nucleic acids, and the like.

The present invention also provides a method for controlled release of an agent. In one embodiment, the invention provides a method for controlled drug release. In one embodiment the method comprises the steps of administering an effective amount of a composition comprising microcarrier and at least one therapeutic agent to a subject, and administering an effective amount of microwave irradiation to a tissue of the subject, wherein the microcarrier releases the at least one therapeutic agent to the irradiated tissue.

The localized and conditional release of the agent allows for a method comprising the delivery of harsh agents that would be harmful to the subject if not delivered at specific locations and conditions. For example, traditional application of some agents could be harmful for the subject, as the agent may harm vital tissues and organs. However, localized and enhanced delivery, when triggered, such as that achieved by the method of the invention, allows for delivery of the same agent because only the irradiated tissues or locations are targeted, while tissues or locations not exposed to microwave irradiation are spared.

Compositions

The present invention provides a composition comprising a microcarrier to provide controlled delivery of an agent when triggered. The microcarrier is a core-shell structured drug microcarrier which comprises a conductive or microwave sensitive core and a thermoresponsive shell. As discussed elsewhere herein, the conductive material produces heat under microwave irradiation thereby heating up the thermoresponsive material to release the agent. In one embodiment, the microcarrier is microwave irradiation-activated, where the embedded therapeutic agent is delivered at a location receiving microwave irradiation. In one embodiment, the microcarrier comprises a core and a shell. In one embodiment, the core is a conductive core or a microwave-sensitive core. In one embodiment, the shell is a thermoresponsive shell. In one embodiment, the microcarrier comprises a polymer on the surface.

In some embodiments, the core comprises a conductive material. For example, in some embodiments, the conductive material is an organic conductive material, inorganic conductive material, or a conductive polymer. Non-limiting examples of conductive materials include poly(p-phenylenediamine), polyaniline, polypyrrole, poly(3,4-ethylenedioxythioxythiophene) (PEDOT),

polyvinylpyrrolidone, and the like. In one embodiment, the core comprises more than one conductive material. In one embodiment, the core comprises additives that modulate the conductive properties of the conductive material. Exemplary additives include, but are not limited to, magnetic ferrites, magnetic metals, transition metal oxides, carbon nanotubes, graphene, activated charcoal, and small molecules such as camphor- 10-sulfonic acid, dinonylnaphthalenedisulfonic acid,

dodecylbenzenesulfonic acid, and the like. In one embodiment, the conductive material is microwave sensitive. In one embodiment, the conductive material is poly(p-phenylenediamine) (PpPD).

In some embodiments, the shell comprises a thermoresponsive material. For example, in some embodiments, the thermoresponsive material is organic heat-sensitive material, inorganic heat-sensitive material, or a heat-sensitive polymer. Non-limiting examples of thermoresponsive materials include poly(N- isopropylacrylamide), poly(N,N-diethylacrylamide), poly(acryloyl piperidine), poly(N-ethylmethacrylamide), poly(N-N-propylacrylamide), poly(N-vinyl pyrrolidone), poly(ethyl oxazoline), poly (N-vinylisobutylamide) (PNVIBA), poly(2- carboxyisopropylacrylamide) (PCIPAAm), poly(N-(3'-methoxypropyl)acrylamide), poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA), hydroxypropyl cellulose, hydroxypropyl methylcellulose, poly(vinylcaprolactame), polyethylene oxide, polypropylene oxide, polyhydroxyethylmethacrylate, poly(methacrylic acid), poly(oxyethylene vinyl ether), poly(hydroxypropyl acrylate) polyvinyl methyl ether, triblock co-polypeptides consisting of short leucine zipper end blocks, elastin-like polypeptides, and combinations and co-polymers thereof. In some embodiments, the thermoresponsive material is a block co-polymer comprising poly(N- isopropylacrylamide). In some embodiments, it may be advantageous to functionalize the end(s) of the polymer chain, such as with a carboxylic acid, an N- hydroxysuccinimide (NHS), an amine, or a maleimide. In one embodiment, the thermoresponsive material is a co-polymer. In one embodiment, the thermoresponsive material is cross-linked, such as via the addition of a diacrylamide such as Ν,Ν'- methylenediacrylamide. In some embodiments, the degree of crosslinking of the thermoresponsive material can be optimized for the intended application by varying the ratio of monomers. In one embodiment, the thermoresponsive material is poly(N- isopropylacrylamide) (PNIPAM). In one embodiment, the thermoresponsive material is a co-polymer of PNIPAM and methacrylic acid. In one embodiment, the thermoresponsive material is a co-polymer of PNIPAM and Ν,Ν'- methylenediacrylamide.

In some embodiments, the composition comprises a stabilizing agent. In one embodiment, the stabilizing agent allows the microcarrier to retain its load at body temperature. In one embodiment, the stabilizing agent modifies the lower critical solution temperature (LCST). In one embodiment the stabilizing agent increases the LCST to be above body temperature. In one embodiment the stabilizing agent increases the LCST to be in the range of about 38°C to about 45°C. In one embodiment, the stabilizing agent is comprised in the shell of the microcarrier. In one embodiment the stabilizing agent includes, but is not limited to, poly(l,3-diene), poly(a-methylstyrene), halogenated olefins, poly(vinylesters), poly(acrylonitrile), poly(methacrylonitrile), poly(N-vinyl carbazole), polyethyleneimine (PEI), poly(sodium styrene sulfonate) (PSS), polydimethylsiloxane, and polyacrylic acid (PAA). In some embodiments, the stabilizing agent is PEI.

In one embodiment, the microcarrier comprises at least one agent. In some embodiments, the agent is a therapeutic agent, an imaging agent, contrast agent, diagnostic agent, a labeling agent, a detection agent, or a disinfectant.

In one embodiment, the composition comprises a microcarrier comprising at least one imaging agent. Imaging agents are materials that allow the microcarrier to be visualized after exposure to a cell or tissue. Visualization includes imaging for the naked eye, as well as imaging that requires detecting with instruments or detecting information not normally visible to the eye, and includes imaging that requires detecting of photons, sound or other energy quanta. Examples include stains, vital dyes, fluorescent markers, radioactive markers, enzymes or plasmid constructs encoding markers or enzymes. Many materials and methods for imaging and targeting that may be used in microcarriers are provided in the Handbook of Targeted delivery of Imaging Agents, Torchilin, ed. (1995) CRC Press, Boca Raton, Fla.

Visualization based on molecular imaging typically involves detecting biological processes or biological molecules at a tissue, cell, or molecular level.

Molecular imaging can be used to assess specific targets for gene therapies, cell-based therapies, and to visualize pathological conditions as a diagnostic or research tool. Imaging agents that are able to be delivered intracellularly are particularly useful because such agents can be used to assess intracellular activities or conditions.

Imaging agents must reach their targets to be effective; thus, in some embodiments, an efficient uptake by cells is desirable. A rapid uptake may also be desirable to avoid the RES, see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001).

Further, imaging agents preferably should provide high signal to noise ratios so that they may be detected in small quantities, whether directly, or by effective amplification techniques that increase the signal associated with a particular target. Amplification strategies are reviewed in Allport and Weissleder, Experimental Hematology 1237-1246 (2001), and include, for example, avidin-biotin binding systems, trapping of converted ligands, probes that change physical behavior after being bound by a target, and taking advantage of relaxation rates. Examples of imaging technologies include magnetic resonance imaging, radionuclide imaging, computed tomography, ultrasound, and optical imaging.

Microcarriers as set forth herein may advantageously be used in various imaging technologies or strategies, for example by incorporating imaging agents into microcarriers. Many imaging techniques and strategies are known, e.g., see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001); such strategies may be adapted to use with microcarriers. Suitable imaging agents include, for example, fluorescent molecules, labeled antibodies, labeled avidimbiotin binding agents, colloidal metals (e.g., gold, silver), reporter enzymes (e.g., horseradish peroxidase), superparamagnetic transferrin, second reporter systems (e.g., tyrosinase), and paramagnetic chelates. Advantages of microcarriers less than about 100 nm or 50 nm in diameter include for example, the ability of the nanoparticles to be readily delivered and taken up by cells. Compared to imaging agents that are merely conjugated to a targeting molecule, microcarriers can increase signal-to-noise ratio by delivering larger imaging agent loads per uptake event resulting in higher amplification. Many imaging agents may be loaded into a microcarrier having a targeting molecule (e.g., tenascin), which passes into a cell via a single uptake event (i.e., caveolar uptake in the case of nanoparticles of less than about 100 nm or 50 nm). In contrast, only a single imaging agent linked to a targeting molecule would be taken up by the same event. Since the internalization, intracellular transport, and recycling of cell surface receptors often requires significant tumaround time, the resultant direct uptake of signal molecules by a cell is slower than the uptake of signal molecules with a microcarriers.

In some embodiments, the imaging agent is a magnetic resonance imaging contrast agent. Examples of magnetic resonance imaging contrast agents include, but are not limited to, 1,4,7, 10-tetraazacyclododecane-N,N',N"N"'-tetracetic acid (DOTA), diethylenetriaminepentaacetic (DTP A), 1 ,4,7, 10- tetraazacyclododecane-N,N', N",N"'-tetraethylphosphorus (DOTEP), 1 ,4,7, 10- tetraazacyclododecane-N,N',N"-triacetic acid (DOTA) and derivatives thereof (see U.S. Pat. Nos. 5,188,816, 5,219,553, and 5,358,704). In some embodiments, the imaging agent is an X-Ray contrast agent. X-ray contrast agents already known in the art include a number of halogenated derivatives, especially iodinated derivatives, of 5- amino-isophthalic acid.

Clinical imaging is of increasing helpfulness in clinical and research settings, e.g., as reviewed by Acharya et al, Computerized Medical Imaging and Graphics, 19(1): 3-25 (1995). Current uses include laboratory medicine, surgery, radiation therapy, nuclear medicine, and diagnostic radiology. Microcarriers may be loaded with agents that enhance these processes, for example by enhancing contrast, or delivering agents to cells that allow for visualization with such techniques.

In one embodiment, the composition comprises a microcarrier comprising at least one therapeutic agent. In one embodiment the at least one therapeutic agent is contained within the thermoresponsive shell of the microcarrier. In one embodiment the at least one therapeutic agent is contained within the core of the microcarrier. In one embodiment, the therapeutic is a small molecule, a nucleic acid, a polypeptide, or an antibody, a siRNA, a ribozyme, an antisense nucleic acid, an antagonist, an aptamer, a peptidomimetic, or any combination thereof. In one embodiment, the composition comprises a targeting domain that directs the microcarrier to a site. In one embodiment, the site is a site in need of the agent comprised within the microcarrier. The targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, and the like that targets the particle to a site in particular need of the therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the microcarrier specifically binds to a target associated with a site in need of an agent comprised within the microcarrier. For example, the targeting domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target can be a protein, protein fragment, antigen, or other biomolecule that is associated with the targeted site. In certain embodiments, the target (e.g.

antigen) associated with a site in need of a treatment with an agent. In some embodiments, the targeting domain may be co-polymerized with the material comprising the microcarrier. In some embodiments, the targeting domain may be covalently attached to the material comprising the microcarrier, such as through a chemical reaction between the targeting domain and the material comprising the microcarrier. In some embodiments, the targeting domain is an additive in the microcarrier.

Small molecule therapeutic agents

In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art. In one embodiment, a small molecule therapeutic agents comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.

Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art, as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development. In some embodiments of the invention, the therapeutic agent is synthesized and/or identified using combinatorial techniques.

In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a

characterized biological structure ("focused libraries") or synthesized with less structural bias using flexible cores. In some embodiments of the invention, the therapeutic agent is synthesized via small library synthesis.

The small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted, and it is understood that the invention embraces all salts and solvates of the therapeutic agents depicted here, as well as the non-salt and non-solvate form of the therapeutic agents, as is well understood by the skilled artisan. In some embodiments, the salts of the therapeutic agents of the invention are pharmaceutically acceptable salts.

Where tautomeric forms may be present for any of the therapeutic agents described herein, each and every tautomeric form is intended to be included in the present invention, even though only one or some of the tautomeric forms may be explicitly depicted. For example, when a 2-hydroxypyridyl moiety is depicted, the corresponding 2-pyridone tautomer is also intended.

The invention also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms of the therapeutic agents described. The recitation of the structure or name herein is intended to embrace all possible stereoisomers of therapeutic agents depicted. All forms of the therapeutic agents are also embraced by the invention, such as crystalline or non-crystalline forms of the therapeutic agent. Compositions comprising a therapeutic agents of the invention are also intended, such as a composition of substantially pure therapeutic agent, including a specific stereochemical form thereof, or a composition comprising mixtures of therapeutic agents of the invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture. The invention also includes any or all active analog or derivative, such as a prodrug, of any therapeutic agent described herein. In one embodiment, the therapeutic agent is a prodrug. In one embodiment, the small molecules described herein are candidates for derivatization. As such, in certain instances, the analogs of the small molecules described herein that have modulated potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and drug development. Thus, in certain instances, during optimization new analogs are designed considering issues of drug delivery, metabolism, novelty, and safety.

In some instances, small molecule therapeutic agents described herein are derivatives or analogs of known therapeutic agents, as is well known in the art of combinatorial and medicinal chemistry. The analogs or derivatives can be prepared by adding and/or substituting functional groups at various locations. As such, the small molecules described herein can be converted into derivatives/analogs using well known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. Also, the linking atoms or groups can be modified into longer or shorter linkers with carbon backbones or hetero atoms. Also, the ring groups can be changed so as to have a different number of atoms in the ring and/or to include hetero atoms. Moreover, aromatics can be converted to cyclic rings, and vice versa. For example, the rings may be from 5-7 atoms, and may be carbocyclic or heterocyclic.

As used herein, the term "analog," "analogue," or "derivative" is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog can be a structure having a structure similar to that of the small molecule therapeutic agents described herein or can be based on a scaffold of a small molecule therapeutic agents described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative of any of a small molecule inhibitor in accordance with the present invention can be used to treat a disease or disorder.

In one embodiment, the small molecule therapeutic agents described herein can independently be derivatized, or analogs prepared therefrom, by modifying hydrogen groups independently from each other into other substituents. That is, each atom on each molecule can be independently modified with respect to the other atoms on the same molecule. Any traditional modification for producing a derivative/analog can be used. For example, the atoms and substituents can be independently comprised of hydrogen, an alkyl, aliphatic, straight chain aliphatic, aliphatic having a chain hetero atom, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic having one or more hetero atoms, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halo- substituted aliphatics, and the like. Additionally, any ring group on a compound can be derivatized to increase and/or decrease ring size as well as change the backbone atoms to carbon atoms or hetero atoms. Nucleic acid therapeutic agents

In other related aspects, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, or miRNA molecule. In one embodiment, the isolated nucleic acid molecule encodes a therapeutic peptide. In some instances the therapeutic agent is an siRNA, miRNA, or an antisense molecule, which inhibits a targeted nucleic acid. In one embodiment, the nucleic acid comprises a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

In one aspect of the invention, a targeted gene or protein, can be inhibited by way of inactivating and/or sequestering the targeted gene or protein. As such, inhibiting the activity of the targeted gene or protein can be accomplished by using a nucleic acid molecule encoding a transdominant negative mutant.

In one embodiment, siRNA is used to decrease the level of a targeted protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al, 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al, 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al, 2003, Cell, 115: 199-208 and Khvorova et al, 2003, Cell 115:209-216. Therefore, the present invention also includes methods of decreasing levels of PTPN22 using RNAi technology.

In one aspect, the invention includes a vector comprising an siRNA or an antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide. The incorporation of a desired polynucleotide into a vector and the choice of vectors are well-known in the art as described in, for example, Sambrook et al. (2012), and in Ausubel et al. (1997), and elsewhere herein.

In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) therapeutic agents. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleave the shRNA to form siRNA.

In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification of expressing cells from the population of cells sought to be transfected or infected using a the microcarrier of the invention. In other embodiments, the selectable marker may be carried on a separate piece of DNA and also be contained within the microcarrier. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

Therefore, in one aspect, the microcarrier may contain a vector, comprising the nucleotide sequence or the construct to be delivered. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and/or eukaryote- vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

By way of illustration, the vector in which the nucleic acid sequence is introduced can be a plasmid, which is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.

The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al, 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

In one embodiment, the recombinant expression vectors may also contain nucleic acid molecules, which encode a peptide or peptidomimetic.

A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

The recombinant expression vectors may also contain a selectable marker gene, which facilitates the selection of host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrawal et al, 1987, Tetrahedron Lett. 28:3539-3542; Stec et al, 1985 Tetrahedron Lett. 26:2191-2194; Moody et al, 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

In one embodiment of the invention, an antisense nucleic acid sequence, which is expressed by a plasmid vector is used as a therapeutic agent to inhibit the expression of a target protein. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of the target protein.

Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal.

Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U. S. Patent No. 5,023,243).

In one embodiment of the invention, a ribozyme is used as a therapeutic agent to inhibit expression of a target protein. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure, which are complementary, for example, to the mRNA sequence encoding the target molecule. Ribozymes targeting the target molecule, may be synthesized using commercially available reagents (Applied Biosy stems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.

In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

Polypeptide therapeutic agents

In other related aspects, the therapeutic agent includes an isolated peptide that modulates a target. For example, in one embodiment, the peptide of the invention inhibits or activates a target directly by binding to the target thereby modulating the normal functional activity of the target. In one embodiment, the peptide of the invention modulates the target by competing with endogenous proteins. In one embodiment, the peptide of the invention modulates the activity of the target by acting as a transdominant negative mutant.

The variants of the polypeptide therapeutic agents may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post- translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

Antibody therapeutic agents

The invention also contemplates a microcarrier comprising an antibody, or antibody fragment, specific for a target. That is, the antibody can inhibit a target to provide a beneficial effect.

The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain FV molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest. The polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Suitable carriers that may be chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled polypeptide may then be used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

Methods of Use

The invention relates to methods of using the microcarriers, microcarrier compositions, and pharmaceutical compositions of the present invention. In various embodiments, the methods relate to administering controlled release of a therapeutic agent.

The methods are useful for the controlled release of a therapeutic agent. In some aspects, the method comprises administering to a subject an effective amount of a composition comprising microcarrier wherein the microcarrier comprises at least one therapeutic agent, and administering an effective amount of microwave irradiation to the subject, wherein the microcarrier releases the at least one therapeutic agent upon microwave irradiation. In some embodiments, the microwave irradiation is delivered to a target location, such as a target tissue or target organ, wherein the microcarrier releases the at least one therapeutic agent in the target tissue or target organ upon irradiation. In one embodiment, the method comprises administering microwave irradiation at a selected time, wherein the microcarrier releases the at least one therapeutic agent at the selected time.

Methods of delivering microwave irradiation to the subject are not particularly limited. For example, in one embodiment, microwave radiation can be delivered to the tissue using one or more microwave antennas. The microwave radiation can be directed onto the tissue or a specific portion thereof (a "target area") from each of antenna. In certain embodiments, microwave radiation can be delivered to the tissue using one three microwave antennas. In such instances, it will be possible to adjust the directionality of one or more of the antennas so that the microwave radiation emitted therefrom will be directed onto the desired target area, which allows the concentration or focusing of the microwave radiation onto the chosen situs. One method of adjusting the antennas so that each one is focused onto the target area is triangulation, which can be accomplished manually or with the assistance of a machine, such as a computer. Computer-assisted triangulation represents a preferred method of adjusting the three microwave antennas in order to direct the emitted microwave radiation onto the desired target area. Triangulation represents a relatively straightforward operation for modern computers, and the computer-assisted triangulation of three microwave antennas is considered to be within the ability of those skilled in the art.

In one embodiment, the microwave irradiation can be applied percutaneously, i.e., through unbroken skin. Thus, the one or more microwave antennas for use in the current invention can be configured for delivering microwave radiation from outside the subject's body. In other instances, one or more microwave antennas can be delivered into or near the target tissue, and the microwave radiation emitted from the antenna and directed onto the adjacent tissue from within the subject's body. Thus, one or more of the microwave antennas can be configured for delivering microwave radiation within the subject's body, and to this end may comprise a probe. The probe can be a conventional microwave antenna catheter, or can comprise a fiber optic cable. In the former instance, the microwave radiation will be produced by and emitted from the catheter itself; in the latter instance, where the probe comprises a fiber optic cable, the microwave radiation will be produced by an external antenna and transported through the fiber optic cable into the subject and onto the target tissue. The fiber optic transportation of microwaves is effected by techniques known to those skilled in the art.

The present invention contemplates delivering microwave irradiation by a microwave radiation generator capable of generating microwave radiation characterized as having at least one frequency component, the generator being operatively connected to at least one antenna, each of the one or more antennas being capable of transmitting microwave radiation to a tissue region; and, a source of particles, the particles being capable of absorbing at least a portion of the transmitted microwave radiation, and being capable of being placed into or near the tissue region.

In some embodiments, microwave radiation is administered at a preselected frequency. Preferably, the pre-selected microwave radiation frequency will be the resonating microwave frequency, i.e., the microwave radiation frequency at which the microcarrier absorbs a maximum amount of microwave radiation. By using methods known in the art, a composition of the present invention can be subjected to different frequencies of microwave radiation and the relative amounts of microwave radiation absorbed can be determined. Preferably, the microwave radiation selected is the frequency that comparatively results in the greatest amount of microwave radiation absorption. In one embodiment, the pre-selected microwave radiation frequency is characterized as having at least one frequency component in the range of from about 100 MHz to about 500 GHz. In one embodiment, the pre-selected microwave radiation frequency is characterized as having at least one frequency component in the range of from about 500 MHz to about 250 GHz. In other embodiments, the pre-selected microwave radiation frequency is characterized as having at least one frequency component in the range of from about 1 GHz to about 100 GHz.

In some embodiments, the microwave radiation is administered at a pre-selected power. For example, in some embodiments, the microwave radiation is administered at a power less than or equal to 500 W. In one embodiment, the microwave radiation is administered at a power in the range of from about 1 W to about 500 W.

In some embodiments, the microwave radiation is administered for a pre-selected period of time. For example, in some embodiments a period of from about a few minutes up to about a few hours is sufficient. In some embodiments of the present invention, the period of time will be up to 2 hours. In some embodiments, the period of time will be up to 1 hour. In some embodiments of the present invention, the period of time will be up to 30 minutes. In some instances, the period of time will be about 10 seconds to about 1 minute. The microwave radiation is administered as pulses, where at least one pulse is administered to the subject. In one embodiment, each pulse may have a duration of 1 -10,000 microseconds depending on the tissue type, tissue size and geometry etc. The number of pulses administered is not particularly limited. In one embodiment, the number of pulses is within the range of about 1 pulse to about 500 pulses.

The microwave generator for use in the present systems can be selected from commercially-available generators and custom-built machines.

Microwave generators are readily available from various commercial vendors, including microwave generators capable of generating microwaves in the C- and X- band frequencies.

Various off-the-shelf antennas can be used to supply one or more of the recited at least one antenna. In preferred embodiments, each of the at least one antenna(s) is capable of transmitting focused microwave radiation. It is also preferred that each of the at least one antenna is capable of being adjusted in order to direct said focused microwave radiation onto a desired target area. In some embodiments of the current invention, the microwave generator is operatively connected to three antennas. Where three microwave antennas are used, the antennas can be configured to permit adjustment. In such instances, it will be possible to adjust the directionality of one or more of the antennas so that the microwave radiation emitted therefrom will be directed onto the desired target area, which allows the concentration or focusing of the microwave radiation onto the chosen situs. One method of adjusting the antennas so that each one is focused onto the target area is triangulation, which can be

accomplished manually or with the assistance of a machine, such as a computer. The instant systems of providing microwave treatment can further comprise a computer, which can be used to assist triangulation, to acquire images, to process images, to display data (e.g., temperature data, depth data), or for other useful purposes.

The methods comprise the administration of a microcarrier composition by any suitable method known in the art. The methods of administration permit the microcarrier composition to be administered locally to the selected target tissue or to be administered at a specific time. In one embodiment, the method of administration includes injection of a solution or composition containing the microcarrier composition. In one embodiment, the microcarrier composition is administered systemically. In other embodiments, other methods of administration, such as sub-cutaneous injection, may be employed where appropriate.

The microcarrier composition described herein can be incorporated into any formulation known in the art. For example, the microcarrier may be incorporated into formulations suitable for oral, parenteral, intravenous, subcutaneous, percutaneous, topical, buccal, or another route of administration. Suitable

compositions include, but are not limited to, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs. In the method of treatment, the administration of the composition of the invention may be for either "prophylactic" or "therapeutic" purpose. When provided prophylactically, the composition of the present invention is provided in advance of any symptom, although in particular embodiments the invention is provided following the onset of one or more symptoms to prevent further symptoms from developing or to prevent present symptoms from becoming worse. The prophylactic administration of composition serves to prevent or ameliorate any subsequent symptom. When provided therapeutically, the pharmaceutical composition is provided at or after the onset of a symptom. Thus, the present invention may be provided either prior to the anticipated exposure to a disorder- causing agent or disorder state or after the initiation of the disorder.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising one or more microcarrier compositions of the present invention. The relative amounts of the microcarrier, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients. Said compositions may comprise additional medicinal agents, pharmaceutical agents, carriers, buffers, adjuvants, dispersing agents, diluents, and the like depending on the intended use and application.

Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include, but are not limited to, a gum, a starch (e.g., corn starch or pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils, Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, turmeric oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.

Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media such as phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. Suitable carriers may comprise any material which, when combined with the biologically active compound of the invention, retains the biological activity. Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present including, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like, in addition, the pharmaceutical composition of the present invention might comprise proteinaceous carriers, e.g., serum albumin or immunoglobulin, preferably of human origin.

A microcarrier composition may be administered alone, or in combination with other drugs and/or agents as pharmaceutical compositions. The composition may contain one or more added materials such as carriers and/or excipients. As used herein, "carriers" and "excipients" generally refer to substantially inert, non-toxic materials that do not deleteriously interact with other components of the composition. These materials may be used to increase the amount of solids in particulate pharmaceutical compositions, such as to form a powder of drug particles. Examples of suitable carriers include water, silicone, gelatin, waxes, and the like.

Examples of normally employed "excipients," include pharmaceutical grades of mannitol, sorbitol, inositol, dextrose, sucrose, lactose, trehalose, dextran, starch, cellulose, sodium or calcium phosphates, calcium sulfate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEG), and the like and combinations thereof. In one embodiment, the excipient may also include a charged lipid and/or detergent in the pharmaceutical compositions. Suitable charged lipids include, without limitation, phosphatidylcholines (lecithin), and the like. Detergents will typically be a nonionic, anionic, cationic or amphoteric surfactant. Examples of suitable surfactants include, for example, Tergitol® and Triton® surfactants (Union Carbide Chemicals and Plastics, Danbury, Conn.), polyoxyethylenesorbitans, for example, TWEEN surfactants (Atlas Chemical Industries, Wilmington, Del), polyoxyethylene ethers, for example, Brij®, pharmaceutically acceptable fatty acid esters, for example, lauryl sulfate and salts thereof (SDS), and the like. Such materials may be used as stabilizers and/or anti-oxidants. Additionally, they may be used to reduce local irritation at the site of administration.

In at least one embodiment, the composition is formulated in a lyophilized form. In certain embodiments, the lyophilized formulation of the composition allows for maintaining microcarrier structure and achieving remarkably superior long-term stability conditions which might occur during storage or transportation of the microcarriers.

Kits of the Invention

The invention also includes a kit comprising compounds useful within the methods of the invention and an instructional material that describes, for instance, the method of administering the microcarriers and compositions as described elsewhere herein. The kit may comprise formulations of a pharmaceutical composition comprising the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. The kit may comprise injectable formulations that may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. The kit may comprise formulations including, but not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a kit, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to administration of the reconstituted composition. The kit may comprise pharmaceutical compositions prepared, packaged, or sold in the form of a sterile aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic diluent or solvent, such as water or 1,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides. Other formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system.

In certain embodiments, the kit comprises instructional material.

Instructional material may include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the device or implant kit described herein. The instructional material of the kit of the invention may, for example, be affixed to a package which contains one or more instruments which may be necessary for the desired procedure. Alternatively, the instructional material may be shipped separately from the package, or may be accessible electronically via a communications network, such as the Internet.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Example 1 : Self- Actuating Polymeric Core-Shell Microcarriers under Microwave Irradiation for High-Efficiency Controlled Drug Release

Described herein is the design of a first-of-its- kind polymeric drug microcarrier based on poly(p- phenylenediamine) (PpPD)/ poly(N- isopropylacrylamide) (PNIPAM) core-shell structured particles for microwave triggered drug release with exceptional high efficiency. The PpPD particle core efficiently absorbs microwave irradiation and converts the electromagnetic radiation to thermal energy, thus actively heating up the PNIPAM shell. Meanwhile the PNIPAM shell stores drug molecules and releases them when heated, achieving a self-actuating behavior for drug release. The controlled release tests for folic acid and etoposide demonstrate that the core-shell polymeric system could serve as a general drug carrier design for highly efficient microwave triggered targeted drug release. Further description of the data presented herein can be found in Shi et al, Journal of Materials Chemistry B, 2017, 5 : 3541 -3549, which is incorporated herein by reference in its entirety.

The materials and methods employed in these experiments are now described.

Preparation of PpPD/PNIPAM core-shell microparticles The synthesis of poly(p-phenylenediamine) (PpPD) microparticles has been described in the literature. In a typical synthesis process, 1 g of

polyvinylpyrrolidone (PVP) and 1 g of p-phenylenediamine (PDA) monomer were dissolved in 10 mL and 20 mL deionized (DI) water under ultrasonication, respectively. Then 1 mL of PVP solution and 3 mL of p-PDA solution were mixed together and diluted to 10 mL with DI water. 0.2 mL of AgNCb solution (0.02 M) was added as the oxidant and the solution turned to dark purple in 5 minutes, indicating the polymerization of monomer for PpPD. After reaction overnight, the product was centrifuged and washed by DI water for several times and re-dispersed in 10 mL DI water. To synthesize the poly(N-isopropylacrylamide) (PNIPAM) shell, 0.1 g of N- isopropylacrylamide monomer, 2 mg of N', N'-methylene-bisacrylamide acting as crosslinker and \ 0 μΐ. of N, N, N', N'-tetramethylenediamine acting as accelerator were added into the suspension of PpPD. After purging with nitrogen gas for 5 minutes, 2 mg of ammonium persulfate (APS) was added into the mixture and the polymerization was carried out for 12 hours. The obtained product was centrifuged and washed by DI water for several times and collected for further characterizations and tests.

Preparation of SiC /PNIPAM core-shell microparticles 10 mg of SiC microparticles are dispersed in 10 mL of DI water. Then

0.1 g of the N-isopropylacrylamide monomer and 2 mg of N,N- methylenebisacrylamide were added to the suspension. After purging with nitrogen gas for 5 minutes, 2 mg of APS was added to the mixture and the polymerization was carried out for 12 hours. The obtained product was centrifuged and washed with DI water several times and collected for further characterization and tests.

Characterization

The morphology and microstructure of samples were observed by using Scanning Electron Microscopy (SEM) (S5500, Hitachi) operating at 5 kV. The Fourier transform infrared spectroscopy (FTIR) spectra of different samples were recorded by the Fourier Transform Infrared Spectrometer (Thermo Mattson, Infinity Gold FTIR) equipped with a liquid nitrogen cooled narrow band MCT detector, using attenuated total reflection cell equipped with a Ge crystal. Before testing, the samples were dried and mixed with KBr powers, and then compressed to slice. The samples were characterized by X-ray diffractometer (XRD) (Rigaku MiniFlex 600) using Cu Ka radiation (λ = 1.5406 A). The UV-vis absorptions of samples from wavelength of 350 to 800 nm were recorded by using UV-vis spectrometer (Evolution 300, Thermo Scientific). A thermogravimetric analyzer (TGA) (TGA 4000, Perkin Elmer) was used to study the degradation behaviors of different samples in non-isothermal conditions. The samples were heated up from 40 to 900 °C at a heating rate of 5 °C min^"1 in air. The real and imaginary parts of the complex permittivity and permeability of PpPD were obtained using a ΡΝΑ-Χ Network Analyzer working in the 2-18 GHz band.

Preparation of samples for cytotoxicity test

Certain amount of PpPD/PNIPAM microparticles were washed by water for several times and then dissolved in the PBS buffer solution. The final sample solutions with certain concentrations were prepared by adding certain amount of PBS buffer solution. Cell culture and cytotoxicity assay

The HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum for 3 days in a humidified incubator at 37°C in an atmosphere of 5% CO2 prior to the cytotoxicity experiments. A standard cell counting Kit-8 (CCK- 8) assay was used for the cytotoxicity assay. The microphage cells were seeded into 96-well plates at a density of 10,000 per well in 100 media and grew overnight. Then, lOuL different concentrations of PpPD/PNIPAM microparticles (from 20 to 100 μg/mL) in DMEM medium and the plate was incubated for 18 hours in the incubator at 37°C and 5% CO2. Then, 10 of CCK-8 solution was added into each well of the plate, which was incubated for another 2 hours at 37 °C and 5% CO2. The absorbance of each sample at 450 nm was measured using a microplate reader (infinite, M200 pro). The cell viability was calculated as the ratio of absorbance of sample well to that of control well (no microparticles). The standard deviation of each measurement was calculated from three independent experiments.

Microwave heating tests

The microwave heating tests were first conducted in aqueous conditions. 3 mg of PpPD/PNIPAM microparticles were dispersed in 1 mL of DI water at room temperature. Then the suspension was irradiated by microwave with frequency of 2.4 GHz, together with another 1 mL of DI water which was set as the controlled sample. The microwave source is a Monowave 300 Microwave synthesis reactor from Anton Paar Instruments. The temperature was measured every 10 seconds and the total irradiation times were 50 seconds and 30 seconds for low power test (120 W) and high power test (360 W), respectively. The error bar was calculated based on three repeating tests.

The microwave heating tests were conducted in dry conditions. 3 mg of dried PpPD/PNIPAM microparticles were dispersed in a vial at room temperature. The vial of sample was irradiated by microwave with frequency of 2.4 GHz, together with another empty vial for 10 seconds. The optical and temperature mapping images of two vials were obtained by Fluke IR thermometer.

Controlled drug release behavior of PpPD/PNIPAM microparticles

Folic acid and etoposide were adopted as drug models to investigate the controlled drug release behavior of PpPD/PNIPAM microparticles. Before releasing test, the PpPD/PNIPAM microparticles were dried and then swollen in solutions of drug models to load drug molecules. The concentrations for folic acid and etoposide solutions were 5 mg/mL and 0.3 mg/mL, respectively. To estimate the loaded amount of drug solution, 10 mL of the drug solution was prepared and then 10 mg of dry PpPD/PNIPAM particles were added to the solution. After the carriers were fully swollen, they were collected by centrifugation. The remaining drug solution was measured to be around 8 mL. About 0.2 mL of the drug solution could be loaded into every 1 mg of PpPD/PNIPAM microparticles. The loading capacities in the

PpPD/PNIPAM microcarrier are 1 g g^_1 for folic acid and 0.06 g g^_1 for etoposide, respectively. The microcarrier also shows high loading efficiency and the drugs can be absorbed into the PNIPAM shell within 1 min since PNIPAM shows a high swelling rate at the initial stage in low-temperature water.

For the test with water bath under microwave as stimuli, 3 mg of PpPD/PNIPAM microparticles were dispersed in 1 mL of DI water and kept at 37 °C for 10 minutes. After the first 10 minutes, the suspension was centrifuged and the initial released amount of drug was tested by UV-vis measurement (for every UV-vis measurement, the volume of drug solution was always manipulated to 1 mL). Another 1 mL of DI water at 37 °C was added. Then the suspension was manipulated with microwave irradiation and stirring without microwave irradiation as a cycle. For each cycle, the sample was firstly irradiated by microwave with power of 120 W for 10 seconds and then centrifuged for UV-vis test. Before it was stirred for 10 minutes at 37 °C in DI water without microwave irradiation, 1 mL of DI water was added. After the non-irradiation stage, the suspension was also centrifuged to test the released amount of drugs and 1 mL of DI water at 37 °C was added before next stage. The test cycle was performed three times

For the test without water bath under microwave as stimuli, 3 mg of PpPD/PNIPAM microparticles were dispersed in 1 mL of DI water and kept at 37 °C for 10 minutes. After the first 10 minutes, the suspension was centrifuged and the initial released amount of drug was tested by UV-vis measurement. The water was further removed by using wipes. Then the sample was manipulated with microwave irradiation and stirring without microwave irradiation as a cycle. For each cycle, the sample was firstly irradiated by microwave with power of 120 W for 10 seconds and then 1 mL of DI water was added to dissolve the released drug and the suspension was centrifuged for UV-vis test. Before it was stirred for 10 minutes at 37 °C in DI water without microwave irradiation, 1 mL of DI water was added. After the non- irradiation stage, the suspension was also centrifuged to test the released amount of drugs, and remaining water was carefully removed before next stage. The test cycle was performed three times

For the test with hot water as stimuli, 3 mg of PpPD/PNIPAM microparticles were dispersed in 1 mL of DI water and kept at 37 °C for 10 minutes. After the first 10 minutes, the suspension was centrifuged and the initial released amount of drug was tested by UV-vis measurement. Then 1 mL of DI water at 42 °C was added. Then the suspension was manipulated with hot water and stirring with 37 °C water. For each cycle, the sample was firstly kept in hot water at 42 °C for 10 seconds and then centrifuged for UV-vis test. Before it was stirred for 10 minutes at 37 °C in DI water without microwave irradiation, 1 mL of DI water at 37 °C was added. After the cool water bath stage, the suspension was also centrifuged to test the released amount of drugs and 1 mL of DI water at 42 °C was added before next stage. The test cycle was performed three times

For the last controlled test, the PpPD/PNIPAM microparticles were kept in 1 mL of DI water at 37 °C for the whole test. For every 10 minutes, the suspension was centrifuged and UV-vis measurement was conducted. Then another 1 mL of DI water at 37 °C was added.

The results of the experiments are now described.

Described herein is the design of a novel core-shell structured polymeric drug microcarrier in which conductive polymer poly(p-phenylenediamine) (PpPD) acts as core and poly(N-isopropylacrylamide) (PNIPAM) serves as shell.

PpPD can effectively absorb microwave irradiation and generate local thermal field in the hybrid carrier system, thus heating the outer PNIPAM layer up. Uniform particles of PpPD could be facilely synthesized at room temperature by employing

polyvinylpyrrolidone (PVP) as surfactant and these particles could be chemically modified to initiate the polymerization of second polymer on their surfaces (Wang, et al., Adv. Funct. Mater., 2008, 18: 1105-1 11 1 ; Min, et al, J. Mater. Chem., 201 1, 21 :6683-6689; Huang, et al, Chem. Eur. J., 2006, 12:4341 -4350). In this drug carrier system, drugs could be stored in the PNIPAM layer through a simple yet controllable deswell-reswell process (Bae, et al., Chem., Rapid Commun., 1987, 8:481 -485). And under microwave irradiation, the heat generated by conductive polymer core could trigger the release of drug through the phase transformation of PNIPAM at high temperature (Figure 1), which is a self-actuating behavior. The polymeric core-shell design ensures that each component functions independently, and synergizes their features (the PNIPAM shell utilizes the heat from PpPD core, as well as preventing heat leakage), thus endowing the drug carrier high efficiency, sensitivity and controllability. The designed microcarrier could release high amount of stored drugs after being exposed to stimuli for only seconds, much more effective than other responsive systems (Table 1). The all-organic design also provides excellent biocompatibility and potential for further chemical modification. Owing to diffusion- controlled drug storage and release mechanisms, this hybrid organic material could serve as a general carrier system for wide-ranging drugs.

Table 1. Composition of drug releasing efficiency of different stimuli-responsive drug

Synthesis and characterization of PpPD/PNIPAM core-shell microparticles

The PpPD/PNIPAM core-shell microparticles were prepared by a two- step polymerization method. The morphologies and microstructures of PpPD and PpPD/PNIPAM core-shell microparticles were investigated by scanning electron microscopy (SEM). Figure 2A shows the SEM images of PpPD micro-crystals. The PpPD particles exhibited a diamond-like shape and relatively uniform size dispersion with length of ~1 μπι, width of ~400nm and thickness of ~400nm. The surface of PpPD particles was smooth and their edges were sharp (inset in Figure 2A), indicating their high crystalline nature. The PpPD/PNIPAM core-shell microparticles were obtained by polymerizing and crosslinking of PNIPAM hydrogel layer in the suspension of pre-synthesized PpPD particles. After being coated by PNIPAM, the hybrid material maintained the diamond- like shape as shown in Figure 2B. However different from the smooth surface of PpPD particles, the surface of hybrid particles became rough and an outer layer with highly porous structure was formed as revealed by the side view image (Figure 3). The above evidences demonstrated the successful synthesis of core- shell microstructured particles. To further confirm the core-shell structure of PpPD/PNIPAM microparticles, STEM imaging was performed to examine both PpPD and PpPD/PNIPAM particles. As shown in Figure 2C, pure PpPD particles were not transparent in the STEM image due to their relatively high thickness. For PpPD/PNIPAM microparticles, an outer layer with thickness of -50 nm could be observed (Figure 2D). This porous layer could be attributed to the formation of crosslinked PNIPAM hydrogel shell with amorphous nature.

The chemical structures of as-synthesized PpPD and PpPD/PNIPAM microparticles were further analyzed by the Fourier transform infrared spectroscopy (FTIR) as shown in Figure 4A. In the spectrum of the pure PpPD particles, the peaks at 3465, 3420, and 3338 cm^"1 correspond to the N-H stretching vibration of the -NH- group, whereas the weak band at 3035 cm^"1 is due to the aromatic C-H stretching vibration. Two sharp peaks could be found at 1604 and 1540 cm^"1 which can be ascribed to the C-N and C-C stretching vibrations of phenazine rings, respectively. The peak at 1502 cm^"1 is induced by the stretching of the benzene ring while two peaks at 1274 and 1236 cm^"1 are associated with the C-N stretching mode in the benzenoid and quinoid imine units (Qiu, et al, J. Phys. Chem. C, 2014, 118: 14929- 14937). All these characteristic peaks could be used to identify the polymerization of p-phenylenediamine (PDA) monomers to PpPD particles. The molecular structure of PpPD particles was also confirmed by Raman spectra (Figure 5). The band between 1510 cm^"1 and 1594 cm^"1 may be ascribed to the N-H bending deformation mode and the C-C deformation of benzenoid rings and quinoid rings (Qiu, et al, J. Phys. Chem. C, 2014, 118: 14929-14937). In the FTIR spectrum of PpPD/PNIP AM microparticles, all the characteristic peaks associated with PpPD could still be observed and a new peak at 2970 cm^"1 could be found which should be ascribed to the -CH₃ asymmetric stretching of PNIPAM. The FTIR test confirmed the co-existence of PpPD and PNIPAM in the hybrid particles.

The composition and crystallinity of as-prepared samples were investigated by X-ray diffraction (XRD). As shown in Figure 4B, the PNIPAM only exhibited a broad band in the low angle side (15°-30°), indicating its amorphous structure. The PpPD particles showed different XRD pattern from that of PDA monomers (Figure 6) with strong and sharp characteristic peaks at 14°, 20°, 21 °, 22°, 28°, 38°, 43° and 64°, indicating that the PpPD were highly crystallized. The XRD spectrum of PpPD/PNIP AM microparticles exhibited both features of PpPD particles and PNIPAM hydrogel. A rising background could be found in the low angle range, which is due to the existence of amorphous PNIPAM. The characteristic peaks associated with PpPD could be observed in the spectrum of PpPD/PNIP AM microparticles and their positions were identical with those of pure PpPD particles, confirming that the crystal structure of PpPD was maintained after hybridizing with the second polymer. However, the intensity of the characteristic peaks in the spectrum of PpPD/PNIP AM microparticles significantly decreased, especially for the peaks at 38°, 43° and 64°. This could be explained by the formation of core-shell structure that the PpPD particles were wrapped by the PNIPAM shell layer (Lu, et al., Polymer Composites, 2009, 30: 847-854; Keplinger, et al., Nano Lett., 2009, 9: 1877-1882). The PNIPAM shell weakened the X-ray irradiated on the PpPD core and the interface between PNIPAM and PpPD may affect the interactions between X-rays and the PpPD crystals, thus resulting in a change in the intensity of the characteristic peaks. The influence of core-shell structure on optical properties of microparticles was also reflected in the UV-vis spectra (Figure 4C). It can be seen that the broad absorption band of PDA monomer around wavelength of 450 nm shifted to ~ 550 nm after the polymerization, owing to the formation of conjugated chains (Sivakumar, et al., Synth. Met, 2002, 126: 123-135; Zimmermann, et al., Synth. Met, 1998, 93 : 17- 25). After the polymerization of PNIPAM, the absorption of hybrid particles was enhanced in the wavelength range of 550-650 nm, due to the scattering of light in the PNIPAM shell. The results of XRD and UV-vis tests further confirmed the core-shell structure of the PpPD/PNIPAM microparticles.

Thermogravimetric analysis (TGA) was used to monitor thermal degradation behavior of different samples and estimate the composition of

PpPD/PNIPAM microparticles (Figure 4D). Before polymerization, PDA monomers showed a low decomposition temperature starting from 150 °C (Figure 7). The initial weight loss appeared around 100 °C for both PNIPAM and PpPD, which was related to removal of solvent, residual monomers and water during the dehydration of surface hydroxyl groups (Wang, et al, J. Mater. Sci., 2012, 47:5918-5925). PNIPAM showed a significant weight loss in the range of 350 to 430°C, after which only 25% of the initial mass remained. This may be attributed to the decomposition of PNIPAM chains. Compared to PNIPAM, PpPD particles exhibited better thermal stability that they maintained 84% of the initial mass at 430°C. The decomposition of PpPD chains started from 520°C and only 7% of the initial mass remained when the temperature reached 625°C. The core-shell PpPD/PNIPAM microparticles presented the thermal degradation behavior that clearly combines the characteristics of both PNIPAM and PpPD. After the initial weight loss related to the removal of moisture and residual monomers, the thermal decomposition of the PpPD/PNIPAM microparticles started from 350 °C (which is the same as PNIPAM) and maintained a relatively constant decomposition rate until 625 °C, after which only 4% of the initial weight was preserved. According to the TGA test, the weight ratio of PNIPAM in the hybrid particles could be estimated to be -44%.

To further evaluate the cytocompatibility of the PpPD/PNIPAM microparticles, HEK293 cells were used to conduct cytotoxicity tests. The cytotoxicity was examined through testing the cell viability upon exposure to the hybrid microparticles by using the standard cell counting Kit-8 (CCK-8) assay. No apparent reduction in cell viability was found after the incubation of the cells with the hybrid microparticles even at a concentration of 100 μg mL ¹ (Figure 8), suggesting that the system has low cytotoxicity. Microwave absorption and thermal conversion properties of PpPD/PNIP AM core- shell microparticles

The microwave absorption and thermal conversion properties are the key features which determine the performance of the hybrid particles in controlled drug release application. Efficient absorption and rapid conversion of microwave to thermal energy is essential to ensure the quick and accurate release of drugs in the local position irradiated by microwave. In order to further examine the microwave absorption of PpPD, films of PpPD in paraffin of thickness 4 and 5 mm were prepared. The reflection loss of the resulting films in the 2-18 GHz band was examined in order to test the real and imaginary parts of complex permittivity and permeability of these PpPD-containing films (Figure 14). Compared to PANI, which is regarded as a typical organic microwave absorption material, PpPD shows slightly lower reflection loss values at high frequency range but similar behavior at low frequency rage. The results demonstrate that PpPD exhibits similar microwave absorption ability to PANI, especially at low frequency ranges.

To demonstrate the excellent microwave thermal response property of PpPD/PNIP AM microparticles, microwave heating tests were performed at room temperature in both aqueous and dry conditions. The frequency of microwave applied was carefully set as 2.4 GHz, which was just in the range for biomedical applications. Since the controlled drug release would be conducted in body environment in practical application, the microwave thermal response behaviors were tested and compared for three samples including pure DI water, aqueous suspension of PpPD microparticles and aqueous suspension of PpPD/PNIP AM microparticles. The time- dependent temperature curves of different samples under low power (120 W) and high power (360 W) of microwave irradiation are shown in Figure 9A and Figure 9B, respectively. It is clear that compared to DI water, both PpPD and PpPD/PNIP AM microparticles exhibited good heating effect under microwave irradiation with power of 120 W. The initial temperature was 25°C, and it could reach 69°C and 62°C in 50 seconds for PpPD and PpPD/PNIP AM microparticles respectively, while pure DI could only be heated to 51°C. More importantly, the temperature of PpPD and

PpPD/PNIP AM microparticles suspensions was increased by 17°C and 15°C within first 10 seconds irradiation while DI water was just heated up by 7°C, demonstrating the efficient microwave absorption and thermal conversion properties of PpPD based materials. The PpPD and PpPD/PNIP AM microparticles showed more advantageous features in microwave thermal response behavior when the power of microwave irradiation was further enhanced to 360 W. The suspensions of PpPD and

PpPD/PNIPAM microparticles could be heated up to 87°C in 30 seconds while DI water can only reach 63°C. The microwave heating tests for PpPD/PNIPAM microparticles suspension demonstrated that PpPD/PNIPAM microparticles could absorb microwave and convert it to thermal energy efficiently and timely in aqueous environment, which laid the foundation for microwave triggered controlled drug release. The thermal conversion experiments were also conducted in PBS buffer solution (Figure 12A and Figure 12B), and the results are similar to those obtained in DI water-based experiments. The microwave heating tests for the PpPD/PNIPAM microparticle suspension demonstrated that PpPD/PNIPAM microparticles could absorb microwave energy and convert it to thermal energy quickly in an aqueous environment, which laid the foundation for microwave triggered controlled drug release.

To eliminate the influence of water, microwave heating test for

PpPD/PNIPAM microparticles were also performed in dry state. The as-prepared PpPD/PNIPAM microparticles were dried in oven first and cooled down to room temperature. Then the particles were moved into a vial and irradiated by microwave with power of 120 W for 10 seconds. As a controlled sample, an empty vial was irradiated by microwave at the same time. The optical and temperature mapping images obtained by Fluke IR thermometer were shown in Figure 9C and Figure 9D. The temperature of vial with PpPD/PNIPAM microparticles was much higher than those of empty vial and background. When comparing the optical and IR image, it should be noted that the temperature distribution was closely related to the distribution of PpPD/PNIPAM microparticles. The position where more

PpPD/PNIPAM microparticles sit exhibited higher temperature. The highest temperature which was as high as 100°C was obtained at the bottom of vial. The microwave heating test in dry state supported the conclusion that PpPD/PNIPAM microparticles exhibited good ability for microwave absorption and thermal conversion effect without the assistance of water. The conjugated structure of PpPD chains help transfer the electromagnetic energy into heat and the PNIPAM shell absorbs the generated thermal energy, as well as preventing heat dissipation. Drug release performance of PpPD/PNIPAM core-shell microparticles

The microwave absorption and thermal conversion tests demonstrated that PpPD/PNIPAM microparticles could effectively absorb microwave irradiation and heat up the hybrid material (Figure 9). Then two kinds of drugs were adopted as model systems to confirm the universality of these drug microcarriers: folic acid and etoposide. Folic acid is a common vitamin with charged molecular nature while etoposide is uncharged and serves a good anticancer chemotherapy drug. Before the release test, the PpPD/PNIPAM microparticles were dried and then swollen in solutions of the drug models to load drug molecules. The amount of loaded drug could be controlled by adjusting the concentration of the drug solutions and the swelling time. When the microparticles absorbed the drug solution to saturation, -0.2 mL of the drug solution could be loaded into every 1 mg of PpPD/PNIPAM microparticles. It should be pointed out that certain amount of polyethyleneimine (PEI) was added when loading drugs to tune the lower critical solution temperature (LCST) of PNIP AM shell to 42°C, thus making possible for PpPD/PNIPAM microparticles to retain their load at body temperature (Ma, et al, Angew. Chem. Inter. Ed., 2015, 127:7484-7488).

Figure 10A shows the folic acid release behavior of PpPD/PNIPAM microparticles under different conditions. To simulate the body environment, the microwave triggered release was first tested in aqueous solution. The suspension of microcarriers was initially kept at 37°C for 10 minutes and then subj ected to microwave irradiation and stirring without microwave irradiation as a cycle to evaluate the repeatability and controllability of the release (Qiu, et al, J. Phys. Chem. C, 2014, 118: 14929-14937). For each cycle, the sample was irradiated by microwave with power of 120 W for 10 seconds, and then stirred for 10 minutes at 37°C in DI water without microwave irradiation. The UV-vis measurement data during a typical controlled release test is shown in Figure 1 1. About 60% of folic acid was released during the irradiation stage in the first cycle and only 5% of the drug was released during the non-irradiation stage in the same cycle. Then about 20% and 6% of folic acid was released during the irradiation stages in the second and third cycle and negligible amounts of released drug could be detected during the non-irradiation stages. After the third cycle, almost 98% of loaded folic acid was released. The results showed that PpPD/PNIPAM microparticles exhibited burst release effect for folic acid under microwave irradiation and retained the load when the microwave source was removed, demonstrating that the microcarrier was capable of effectively controlling the folic acid release by adjusting microwave on/off states. The PpPD/PNIPAM microparticles show similar controlled release behavior in PBS buffer solutions when compared to those in DI water (Figure 13). To minimize the heating effect of water, the microwave-triggered release was also tested in dry state. Different from the first test, water was removed by ultracentrifugation and wiped before the particles were irradiated by microwave. The drug release curve was similar to that obtained for first test, but the amount of released drug was slightly lower in each cycle. This could be explained by the diffusion effect of drugs in aqueous environment. The drug release experiments were also conducted in PBS buffer solution.

To further solidify the conclusion that the efficient release of drug for PpPD/PNIPAM microparticles was triggered by microwave, three other control experiments were conducted. For the first control, the folic acid loaded particles were kept at 37 °C in DI water without any irradiation, and it can be seen that only -9% of the drug was released after 40 minutes. For the second control experiment, the stimulus of microwave was replaced by hot water with temperature of 45 °C. The suspension of microcarriers was stirred in hot water for 10 seconds and then kept at 37°C in DI water as a cycle. It was found that only about 5-10% of folic acid was released during the heated stage for each cycle. In the third control experiment (Figure 15), SiC /PNIPAM microparticles (STEM image shown in Figure 16) are used as drug carriers for folic acid and etoposide. The results show that the amounts of drugs released in this control experiment are only slightly higher than those in the control experiment using a hot water bath. The reason is that within each 10 seconds of microwave irradiation, the water is heated from 37 °C to around 45 °C, which is similar to the temperature of the hot water bath. However, since the SiC core is not able to respond to microwave irradiation and generate local heat, the PNIPAM shell can only be heated by the surrounding water, similar to that in the hot water bath. These control experiments demonstrated that the temperature change of the surrounding media had no significant influence on the folic acid release behavior of the PpPD/PNIPAM microparticles. The efficient release of the drugs under microwave irradiation indeed results from the property that the particles can actively absorb microwave energy and the unique design of the core-shell structure in which the inner core can act as the thermal source and heat up the hybrid particles. The drug release efficiency of the present hybrid system was compared to other stimuli-responsive carriers for drug delivery including microwave-, thermal-, light-, and electro-triggered systems and a comparison table is given in Table 1. In most of these works, the drug carriers are prepared by co-polymerization or physical mixture and the drugs are directly loaded into these stimuli-responsive materials. It is challenging for these carriers to simultaneously realize both high responsiveness and fast drug release. Different from previous works, the present system synergizes the advantages of the conjugated polymer PpPD and the PNIPAM hydrogel: PpPD absorbs microwave radiation and efficiently converts it to thermal energy while the PNIPAM hydrogel shows highly responsive sensitivity to a temperature change and thus enables burst drug release. More importantly, the synergic effect between PpPD and PNIPAM is maximized by the core-shell structure design. With this unique structure, PpPD and PNIPAM can function independently without sacrificing their responsive properties. They can further promote each other's functionality since the PpPD core generates local heat and thus acts as an efficient heat source while the

PANIPAM shell absorbs the generated thermal energy and prevents heat dissipation. As a result, the presently described polymeric core-shell structured microparticles show high efficiency, with over 50% of the drugs being released within 10 seconds, which is beneficial for targeted drug release. The stability of the presently described microparticles is also good, as the tested samples show similar releasing performance after being stored in air for two weeks.

Since the folic acid is negatively charged, etoposide was also used as a charge-neutral drug to investigate the controlled drug release property of

PpPD/PNIPAM microparticles. Etoposide shows good pharmacological activity and serves as a good anti-cancer drug which requires targeted release with highly accurate control and efficiency (Williams, et al, N. Engl. J. Med., 1987, 316: 1435-1440; Hande, Eur. J. Cancer, 1998, 34: 1514-1521). The same testing methods were applied for etoposide and the results were shown in Figure 10B. The release tests for etoposide showed a similar trend to those of folic acid that burst release could be triggered by the microwave irradiation, but not by simply changing the temperature of surrounding media. Therefore, the PpPD/PNIPAM microparticles could serve as a general, highly efficient drug carrier system for wide-ranging drugs for targeted drug release triggered by microwave irradiation. In summary, the results presented herein demonstrate the rational design and synthesis of a novel PpPD/PNIPAM core-shell microcarrier for microwave triggered drug release. In this unique design, the PpPD particle core functions as the microwave responsive component which could efficiently convert the electromagnetic radiation to thermal energy and immediately heat up the PNIPAM shell, while the outer shell of PNIPAM could store large amount of drug molecules and bursting release them right up being heated by the PpPD core. The effectiveness of this core- shell structure was proved by release tests for folic acid and etoposide as general drug models, in which the drugs could be efficiently released triggered by microwave irradiation. This work provides a general design of a useful drug carrier for microwave triggered targeted drug release and also demonstrates the important role of architecture of polymeric hybrid material in determining the drug release

characteristics.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A composition for controlled release of an agent, the composition comprising at least one microcarrier having a shell and a core, wherein the microcarrier comprises an effective amount of at least one agent.

2. The composition of claim 1, wherein the shell comprises a thermoresponsive material.

3. The composition of claim 2, wherein the shell comprises the at least one agent.

4. The composition of claim 2 wherein the thermoresponsive material is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), poly[2- (dimethylamino)ethyl methacrylate] (pDMAEMA), hydroxypropylcellulose,

poly(vinylcaprolactame), polyethylene oxide, polyvinylmethylether,

polyhydroxyethylmethacrylate and polyvinyl methyl ether.

5. The composition of claim 2 wherein the thermoresponsive material is PNIPAM.

6. The composition of claim 1, wherein the core comprises a microwave responsive material.

7. The composition of claim 6 wherein the microwave responsive material is selected from the group consisting of poly(p-phenylenediamine) (PpPD), polyaniline and polypyrrole.

8. The composition of claim 6 wherein the microwave responsive material is PpPD.

9. The composition of claim 1, wherein the composition comprises a microwave-responsive element such that the microcarrier releases the at least one agent under microwave irradiation.

10. The composition of claim 1, wherein the at least one agent is selected from the group consisting of at a therapeutic agent, an imaging agent, a diagnostic agent, a contrast agent, a labeling agent or a disinfectant agent.

11. The composition of claim 10, wherein the at least one therapeutic agent is selected from the group consisting of a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, and an antisense nucleic acid molecule.

12. A method for controlling the release of an agent compri administering to a subject an effective amount of a composition comprising a

microcarrier wherein the microcarrier comprises at least one agent, and administering an effective amount of microwave irradiation to the subject, wherein the microcarrier releases the at least one agent after microwave irradiation.

13. The method of claim 12, wherein the microwave irradiation is administered to a target location, wherein the microcarrier releases the at least one agent in the target location.

14. The method of claim 12, wherein the microwave irradiation is administered at a selected time, wherein the microcarrier releases the at least one agent at the selected time.

15. The method of claim 12, wherein the microcarrier comprises a shell and a core.

16. The method of claim 15, wherein the shell comprises a

thermoresponsive material.

17. The method of claim 15, wherein the shell comprises the at least one agent.

18. The method of claim 17, wherein the thermoresponsive material is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), poly[2- (dimethylamino)ethyl methacrylate] (pDMAEMA), hydroxypropylcellulose, poly(vinylcaprolactame), polyethylene oxide, polyvinylmethylether, polyhydroxyethylmethacrylate, and polyvinyl methyl ether.

19. The method of claim 15, wherein the core comprises a microwave responsive material.

20. The method of claim 19, wherein the microwave responsive material is selected from the group consisting of poly(p-phenylenediamine) (PpPD), polyaniline, and polypyrrole.

21. The method of claim 12, wherein the at least one agent is selected from the group consisting of at a therapeutic agent, an imaging agent, a diagnostic agent, a contrast agent, a labeling agent, and a disinfectant agent.

22. The method of claim 21, wherein the therapeutic agent is selected from the group consisting of a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, and an antisense nucleic acid molecule.

23. A method of releasing an agent into a fluid comprising

administering to a fluid an effective amount of a composition comprising a microcarrier wherein the microcarrier comprises at least one agent, and administering an effective amount of microwave irradiation to the fluid, wherein the microcarrier releases the at least one agent after microwave irradiation.