WO2018089918A1 - Système d'administration transdermique à base de micro-aiguilles et son procédé de fabrication - Google Patents

Système d'administration transdermique à base de micro-aiguilles et son procédé de fabrication Download PDF

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Publication number
WO2018089918A1
WO2018089918A1 PCT/US2017/061353 US2017061353W WO2018089918A1 WO 2018089918 A1 WO2018089918 A1 WO 2018089918A1 US 2017061353 W US2017061353 W US 2017061353W WO 2018089918 A1 WO2018089918 A1 WO 2018089918A1
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Prior art keywords
vaccine
cells
microneedle
biodegradable
antigen
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PCT/US2017/061353
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English (en)
Inventor
Martin J. D'souza
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The Corporation Of Mercer University
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Priority claimed from US15/350,718 external-priority patent/US10463608B2/en
Application filed by The Corporation Of Mercer University filed Critical The Corporation Of Mercer University
Priority to EP17869270.3A priority Critical patent/EP3538116A4/fr
Publication of WO2018089918A1 publication Critical patent/WO2018089918A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
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    • A61K39/095—Neisseria
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    • A61K39/12—Viral antigens
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
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    • B29C39/00—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor
    • B29C39/02—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor for making articles of definite length, i.e. discrete articles
    • B29C39/021—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor for making articles of definite length, i.e. discrete articles by casting in several steps
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B29C39/00—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B29L2031/753—Medical equipment; Accessories therefor
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    • B29L2031/756—Microarticles, nanoarticles
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2710/20071—Demonstrated in vivo effect
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
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Definitions

  • the present disclosure relates, in exemplary embodiments, to a system for delivering bioactive and other materials through the skin (transdermally) using microneedles containing the material.
  • the present disclosure also relates, in exemplary embodiments, to methods for forming biodegradable microneedles containing the bioactive material.
  • Microneedles are micron diameter- sized needles, which upon insertion into the skin result in formation of aqueous conduits forming a passage for the vaccine antigens towards the immune-competent skin layers. Due to their short needle length, they avoid contact with the nerve endings in the dermis thus remain to be a painless mode of immunization.
  • IntanzaTM by Sanofi Pasteur
  • IntanzaTM an intradermal influenza vaccine that incorporates a 1.5 mm needle attached to a pre-filled syringe loaded with flu antigens. It has been shown to be efficacious when compared with an IM flu vaccine thus bringing a switch from hypodermic needles to "micro"-needles for immunizations. This opens a new avenue of vaccine delivery through an effective, painless and patient- friendly route of administration.
  • a transdermal delivery system of microneedles containing a bioactive material comprising at least one layer of a support material, at least one biodegradable needle associated with the support material, each needle comprising at least one biodegradable polymer and at least one sugar, wherein each biodegradable needle is hollow and is adapted to retain a bioactive material.
  • a biodegradable microneedle comprising: at least one biodegradable needle associated with the support material, each needle comprising at least one biodegradable polymer, and at least one sugar; wherein each biodegradable needle is at least partially hollow and is adapted to retain a bioactive material.
  • a method of forming transdermal delivery system comprising: (a) providing at least one biodegradable polymer material; (b) dissolving the polymer material in a solvent to form a solution; (c) mixing the solution of polymer material of step (b) with at least one sugar to form a polymer-sugar mixture; (d) providing a bioactive material; (e) providing a microneedle mold; (f) adding the bioactive material and the polymer- sugar mixture of step (c) to the microneedle mold; and, (g) forming at least one microneedle from the polymer- sugar mixture, the at least one microneedle having at least a portion that is hollow, wherein the bioactive material is retained within the hollow portion of the microneedle.
  • a method of transdermally delivering a bioactive material comprising: (a) forming at least one biodegradable and at least partially hollow microneedle from at least one biodegradable polymer and at least one sugar; (b) associating a bioactive material with the at least one microneedle; (c) associating the at least one microneedle with a backing layer; and, (d) contacting the at least one microneedle containing the bioactive material with the skin of a subject, whereby the at least one microneedle introduces the bioactive material to the subject and the at least one microneedle biodegrades.
  • a method of forming transdermal delivery system comprising: (a) mixing PVA, HPMC, and the at least one sugar in a vessel; (b) dissolving the mixture of step (a) in water to and mixing to form a mixture; (c) adding ammonium hydroxide to the mixture of step (b) and mixing; (d) adding to the mixture of step (c) at least one bioactive material in microencapsulated form to form a formulation; (e) adding an aliquot of the formulation of step (d) to a microneedle mold; and, (f) centrifuging the microneedle mold and formulation of step (e) to force the formulation into the microneedle mold.
  • Fig. 1A is a diagram showing mechanisms of interaction of N. gonorrhoeae with cells of the immune system.
  • Fig. IB is a schematic diagram of a microparticulate delivery system.
  • FIG. 2 is schematic view of a microneedle device according to one exemplary embodiment used for transdermal delivery of particles.
  • Fig. 3A is a scanning electron microscopic (SEM) image of formalin fixed whole cell N. gonorrhea which is the antigen for the vaccine.
  • Fig. 3B is a SEM image of spray dried microparticles containing the gonorrhea vaccine antigen.
  • Fig. 3C is a SEM image of the dissolving microneedles which contain the gonorrhea vaccine microparticles for transdermal delivery.
  • Fig. 4 is a graph of N. Gonorrhea specific IgG antibody level measurements in serum via ELISA.
  • Fig. 5 is a schematic flow diagram of one exemplary embodiment of a solvent casting process for fabrication of microneedles.
  • Fig. 6 A is a photomicrograph of porcine ear skin sample showing microchannels created by a microneedles patch.
  • Fig. 6B is a photomicrograph of porcine ear skin sample showing microchannels created by a microneedles patch.
  • Fig. 7 is a schematic view of in vivo tracking over time (30 min., 3 hours, 6 hours and 24 hours) of a vaccine after delivery to a mouse.
  • Fig. 8 is a SEM image of the microneedles being dissolved in the mice after 12 minutes of application of the patch.
  • Fig. 9 is graph of serum IgG levels over time.
  • Fig. 10A is a graph of CD4 T cell counts of lymph nodes.
  • Fig. 10B is a graph of CD4 T cell counts of spleens.
  • Fig. IOC is a graph of CD8 T cell counts of lymph nodes.
  • Fig. 10D is a graph of CD8 T cell counts of spleens.
  • Fig. 11 is a graph of antigen specific CD4 and CD8 T cells counts.
  • Fig. 12A is a SEM image of polymer microparticle matrix formed.
  • Fig. 12B is a SEM image of breast cancer nano-vaccine particles.
  • Fig. 13 is a graph of induction of nitric oxide release by dendritic cells (DC 2.4).
  • Fig. 14A is a graph of MHC I expression.
  • Fig. 14B is a graph of CD 40 expression.
  • Fig. 14C is a graph of MHC II expression.
  • Fig. 14D is a graph of CD 80 expression.
  • Fig. 15 is a graph of tumor volume measurement.
  • Fig. 16A is a graph of percent cell count measurement of lymph nodes CD 4 T cells.
  • Fig. 16B is a graph of percent cell count measurement of spleen CD 4 T cells.
  • Fig. 16C is a graph of percent cell count measurement of spleen CD 8 T cells.
  • Fig. 16D is a graph of percent cell count measurement of spleen CD 8 T cells.
  • Fig. 17 is a schematic illustration of the structure of influenza virus.
  • Fig. 18 is transmission electron microscopy (TEM) image of microparticles of inactivated influenza virus.
  • Fig. 19 is a schematic flow diagram of one exemplary embodiment of a process for fabrication of microneedles.
  • Fig. 20 is a graph of the measurement of mean fluorescent intensity of solutions and microparticles of various groups.
  • Fig. 21 is a graph of the measurement of mean fluorescent intensity of solutions and microparticles of various groups.
  • Fig. 22 is a graph of the measurement of mean fluorescent intensity of solutions and microparticles of various groups.
  • Fig. 23 is a graph of the measurement of mean fluorescent intensity of solutions and microparticles of various groups.
  • Fig. 24 is a graph of serum titers over time.
  • Fig. 25 is a graph of flow cytometry cell count of CD4 and CD8.
  • Fig. 26 is a schematic illustration of antigens and other materials in a polymer matrix.
  • Fig. 27 is a SEM image of microparticles.
  • Fig. 28 is a graph of nitric oxide concentration of various solutions.
  • Fig. 29 is a graph of mean fluorescence intensity of CD86 and CDE 80.
  • Fig. 30 is a graph of mean fluorescence intensity of CD40 and MHC II.
  • Fig. 31 is a graph of log titer over 32 weeks.
  • Fig. 32 is a graph of cell surface expression.
  • Fig. 33 is a graph of cell surface expression.
  • Fig. 34 is a SEM of vaccine loaded microparticles taken using the Phenom Desktop SEM by placing microparticles on a carbon film and observing at 20kV. Microparticles are irregularly shaped in the size range of 1-2 um.
  • Fig: 35 is a photograph of SDS PAGE of F-VLPs extracted from micro particles. Samples were resolved on a prepared 12% polyacrylamide gel and stained using Coomassie Blue. Lane 1: Molecular weight ladder (kDa); Lane 2: Blank microparticles (MPs); Lane 3: VLP-Suspension and Lane 4: F-VLP extracted from microparticles. The VLP was found to be intact in microparticulate form.
  • Fig. 36 is a graph showing the amount of nitric oxide released ( ⁇ ) from DC 2.4 cells when exposed to Cells Only, Blank MP, RSV F-VLP Suspension, RSV F-VLP MP (*p ⁇ 0.05).
  • Fig. 37 is a graph showing the amount of nitric oxide released from DC 2.4 cells when exposed to VLP Suspension, RSV VLP MP and RSV VLP MP + Alum, MPL A and MF59 (*p ⁇ 0.05) There was a significant release of nitric oxide seen in supernatant of cells receiving RSV F- VLP + Alum/ MF59 MPs.
  • Fig. 38 is a graph showing the expression of CD40 on DC 2.4 cells exposed to Blank MP, F-VLP solution, F-VLP MP and VLP MP with adjuvant. MHC II expression was significantly higher in MP group compared to VLP solution.
  • Fig. 39 is a graph showing the expression of CD80 on DC 2.4 cells exposed to Blank MPs, F-VLP solution, F-VLP MP and VLP MP with adjuvants. CD80 is a co-stimulatory molecule required for activation of CD8 T cells.
  • Fig. 40 is a graph showing the expression of CD40 on DC 2.4 cells exposed to Blank MP, F-VLP solution, F-VLP MP and VLP MP with adjuvant.
  • Fig. 41 is a schematic illustration of a timeline for an animal study.
  • Fig. 42 is a graph showing IgG antibody levels in blood serum of mice inoculated with Inactivated RSV vaccine (FI-RSV), solution form of F-VLP, F-VLP microparticles and F-VLP + MPL microparticles.
  • FI-RSV Inactivated RSV vaccine
  • Fig. 43 is a graph showing body weight measurements of mice 6 days post-challenge with live RSV A2 virus. Untreated mice (PBS) showed the highest change in weight compared to vaccinated mice.
  • Fig. 44 is a graph of (spleen) CD4+ and CD8+ T cell response after challenge with live RSV A2 virus.
  • Fig. 45 is a graph of (lymph node) CD4+ and CD8+ T cell response after challenge with live RSV A2 virus.
  • Fig. 46 is a graph of viral titers measured in lung homogenates of various groups after challenge using RT-PCR.
  • Fig 47 is a schematic flow diagram of one exemplary embodiment of a method used for the preparation of microcapsules encapsulating live pancreatic beta cells using biocompatible polymer.
  • Fig. 48 is a schematic diagram showing a portion of an exemplary embodiment of a method of forming microcapsules.
  • Fig. 49 is a graph of the size of microcapsules (diameter being in ⁇ ) plotted at different gas flow rates. Plotted values are mean with + standard deviation bars.
  • Fig. 50 is a graph of microcapsule size distribution obtained after spraying the alginate suspension at 250 L/Hr.
  • Fig. 51 are microcapsule images taken at 10X after spraying the alginate suspension at 250 L/Hr.
  • Fig. 52 is a graph of FTIR spectra of sodium alginate.
  • Fig. 53 is a graph of FTIR spectra of alginate microcapsules.
  • Fig. 54A is a light microscopic image showing clusters of microcapsules encapsulated beta islet pancreatic cells were taken at magnification of 10X.
  • Fig. 54B is a light microscopic image showing clusters of microcapsules encapsulated beta islet pancreatic cells were taken at magnification and 40X.
  • Fig. 55 is a chart of Live dead cell stained images of microcapsules encapsulated pancreatic islet beta cells collected over thirty days period at magnification of 10X.
  • Fig. 56A is a graph of nitric oxide release vs MC Blank (Microcapsules without beta islet pancreatic cells), MC cells (Microcapsules encapsulate beta islet pancreatic cells and Cells only (Unencapsulated cells) ns-not significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant.
  • Fig. 56B is a graph of nitric oxide release vs MC Blank (Microcapsules without beta islet pancreatic cells), MC cells (Microcapsules encapsulate beta islet pancreatic cells and Cells only (Unencapsulated cells) ns-not significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant.
  • Fig. 57 is graph of short term stability monitored by measuring the fraction of intact microcapsules at different concentration of chitosan used as second layer on alginate microcapsules.
  • Fig. 58 is a graph of long term stability monitored by measuring the fraction of intact microcapsules.
  • Fig. 59 is a graph of blood glucose levels of different groups in mice measured for 35 days.
  • Fig. 60 is a graph of percent graft survival plotted for different groups i.e., Diabetic control, MC cells (Microcapsules encapsulated pancreatic beta islet cells) and Cells only (Unencapsulated cells).
  • Fig. 61 is a graph of fractional weight of mice in different groups measured for 35 days.
  • Fig. 62A is a graph of percentage of CD4 and CD8 positive cells plotted for different groups i.e., Diabetic control, MC beta and naked Cells Only. *p ⁇ 0.05 significant, **p ⁇ 0.01 very significant, ***p ⁇ 0.001 extremely significant (spleen cells).
  • Fig. 62B is a graph of percentage of CD4 and CD8 positive cells plotted for different groups i.e., Diabetic control, MC beta and naked Cells Only. *p ⁇ 0.05 significant, **p ⁇ 0.01 very significant, ***p ⁇ 0.001 extremely significant (spleen cells).
  • Fig. 63 A is a graph of percentage of CD4 positive cells plotted for different groups i.e., Diabetic control, MC beta and naked Cells Only. *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant (Lymph node cells).
  • Fig. 63B is a graph of percentage of CD8 positive cells plotted for different groups i.e., Diabetic control, MC beta and naked Cells Only. *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant (Lymph node cells).
  • Fig. 64 is a graph of a flow cytometric analysis showing CD45R cell counts in different groups of mice *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant.
  • Fig. 65 is a graph of a flow cytometric analysis showing CD62L cell counts in different groups of mice *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant.
  • microneedle systems and methods can be adapted for delivery to or through other structures, such as, but not limited to, blood vessel walls, muscle tissue, organs, and the like.
  • Neisseria gonorrhoeae the gonococcus, or GC
  • the Gc interacts with the immune system and prevents the generation of an adaptive immune response.
  • Gc can interact with various immune cells to elicit innate inflammatory responses and suppress Thl/Th2-mediated specific immune responses (Fig. 1).
  • Phagocytosis by macrophages results in activation of NLRP3 inflammasomes, the production of IL-1 and activation of PMNs, and activation of cathepsin B, which leads to pyronecrosis of APC (Duncan et al., 2009).
  • B Interactions with DCs lead to up-regulation of PDL-1 and PDL-2, which induce apoptosis of cells bearing PD1.
  • IL-10 which has immunoregulatory properties and stimulates type 1 regulatory T cells (Trl) (Zhu et al., 2012).
  • C Interaction with CD4+ T helper cells (or B cells) induces secretion of IL-10, TGF- ⁇ , and IL-6 (Liu, Islam, Jarvis, Gray-Owen, & Russell, 2012).
  • IL-10 and TGF- ⁇ suppress the activation of Thl and Th2 cells both directly, and through the activation of Trl cells.
  • TGF- ⁇ and IL-6 drive the development of Thl7 cells which secrete IL-17 and IL-22, leading to the recruitment or induction of innate defenses such as PMNs and anti-microbial peptides.
  • Gc is able to resist destruction by PMNs and anti-microbial peptides while concomitantly suppressing the development of adaptive immune responses such as Gc- specific antibodies that could enhance phagocytosis and intracellular killing by phagocytes and bacteriolysis through the classical complement pathway (Jerse, Bash, & Russell, 2014).
  • adaptive immune responses such as Gc- specific antibodies that could enhance phagocytosis and intracellular killing by phagocytes and bacteriolysis through the classical complement pathway (Jerse, Bash, & Russell, 2014).
  • the proposed vaccine formulation consists of formalin fixed dead whole cell gonococcal encapsulated in albumin-based microparticles that mimic the chemical conjugation process of CPS to a protein carrier similar to meningitis, and enhance antigen uptake via albumin receptors, and elicit a T-cell-dependent immune response. Further, the proposed vaccine exists as a dry powder form and kept well protected from moisture. Thus, the shelf lives of these vaccines are expected to be several fold higher than that of the conventional vaccines.
  • the novel nanotechnology-based vaccine that mimics conjugation effects by encapsulation into albumin-based nanoparticle matrices provides the following advantages: 1- does not require chemical conjugation, 2- self adjuvanting-antigen delivery vehicle, 3- enhanced uptake by immune cells and slow antigen release, i.e. antigen depot effect, 4- induces robust autophagy formation that enhances antigen presentation, 5- uses a heat- stable formulation that does not require refrigeration, 6- can be administered via microneedles 7- the cost of producing this vaccine is dramatically reduced due to the elimination of the costs of chemical conjugation process, purification and packaging in individual dose ampoules that requires constant cooling with limited shelf life, and 8- can be used to formulate and deliver other bacterial and viral vaccines.
  • Neisseria gonorrhoeae is the main bacteria which causes gonorrhea infections.
  • Microneedle based Transdermal vaccination [00121] Microneedle based Transdermal vaccination:
  • the skin provides a unique site for the vaccination purposes as it is easily accessible and houses various immune cells for an efficient immune response against a range of antigens.
  • Skin serves as a barrier against various pathogens and is equipped with the skin associated lymphoid tissues (SALT) to combat any insult from invading pathogens.
  • SALT skin associated lymphoid tissues
  • Various skin cells assist in generation of effective immune response (Gao, Pan, Chen, Xue, & Li, 2008).
  • Keratinocytes are the most pre-dominant (95%) epidermal cells in the skin. They can be activated by pathogens and result in production of cytokines, which in turn recruits dendritic cells/antigen-presenting cells to the site of action leading to initiation of the immune response.
  • Langerhans cells comprise of only 2% of the total cell population in the epidermis but due to their extended dendrites spread in the epidermal layer they cover over 25% of the skin surface. These are professional phagocytic cells efficient in immune surveillance and further signaling to the T-cells present in their vicinity. Activated macrophages and T-cells drain into nearby lymph nodes leading to an enhanced immune response.
  • most of the vaccines are administered via subcutaneous or intramuscular route. These have been highly effective in generating protective immune response but they remain to be invasive, painful and require a skilled professional for vaccination.
  • Microneedles are micron-sized diameter needles, which upon insertion into the skin result in formation of aqueous conduits forming a passage for the vaccine antigens towards the immune- competent skin layers (Fig. 2). Due to their short needle length, they avoid contact with the nerve endings in the dermis thus remain to be a painless mode of immunization.
  • IntanzaTM by Sanofi Pasteur
  • an intradermal influenza vaccine that incorporates a 1.5 mm needle attached to a pre-filled syringe loaded with flu antigens.
  • Figs. 3A-B shows that the cells were intact and were in their native form.
  • This antigen was mixed with a blend of biodegradable and biocompatible cellulose polymer matrix. This was then spray dried and microparticles were made. These microparticles were characterized for their size, charge etc.
  • Particle counter that works on the principle of laser diffraction. Particle size was measured in triplicates for blank as well as vaccine microparticles. For zeta potential measurement, five micrograms of microparticles were suspended in 1 ml of deionized water and measured using a Malvern Zetasizer. Zeta Potential was measured for blank as well as antigen loaded microparticles in triplicates. The particle size, recovery yield and zeta potential are all shown in Table 1.
  • Microparticles may be formed according to the method disclosed in U.S. Patent
  • a bioactive material e.g., whole cell lysate (WCL)
  • CPD cellulose acetate phthalate
  • HPMCAS hydroxyl-propyl methylcellulose acetate succinate
  • Trehalose 5% w/w.
  • microparticles including an adjuvant can be prepared following the same procedure.
  • particles may be made with adjuvant loading of 2.5% w/w.
  • the average particle size may be in a range of about
  • any of a variety of different sugars can be uses, including, but not limited to, trehalose, maltose, sucrose, or the like.
  • Dissolving microneedles intended for the painless transdermal release of encapsulated pharmaceutical agents after dermal insertion, were developed as a solution to the safety issue.
  • Dissolvable microneedles mainly deploy PDMS micromolds which are made from a master structure of microneedles (Fig. 5). Briefly, Polydimethylsiloxane (PDMS) (Dow Chemicals) was poured onto the stainless steel master structure (Step 1-3; Fig. 5). The microneedles were made using the following formula:
  • the maximum speed is 2000 rpm which is achieved step wise, in order to avoid jerk in the rotation process, time for which centrifugation should be done is 5-10 min. Speed should be lowered gradually for same reason as above.
  • the backing layer may be composed of the same components as the microneedle matrix as described in various embodiments herein, however, the backing layer is formed without any microparticles. Keep the molds in tubes and place it in an incubator at 37°C overnight (Step 7-9; Fig. 5).
  • the microneedle patch is 1 cm x 1 cm in size which contains 100 microneedles (10 x 10). The microneedles are 600 ⁇ .
  • mice (Balb-C) via subcutaneous route which serve as conventional routes of administration of vaccines.
  • There were 3 groups, subcutaneous microparticle vaccine (GnH MP), subcutaneous vaccine suspension (GnH Susp) and negative control which was received the blank microparticles (n 6).
  • Blood samples were collected prior to prime dose followed every 2 weeks.
  • the antibody levels in the blood were measured using specific ELISA by plating the antigen on the plate.
  • the antigen specific antibody in the serum was detected using ELISA.
  • a rise in specific antibody levels is seen in Fig. 4 in groups which received the vaccine when compared to the blank microparticles which serve as the negative control after week 4.
  • Fig. 10A-D show the CD4 cell counts of lymph nodes (Fig. 10A) and spleens (Fig. 10B) and the CD8 cell counts in lymph nodes (Fig. IOC) and spleens (Fig. 10D).
  • the groups which received the Gonorrhea vaccine microparticles incorporate in the microneedles showed significantly higher levels of both CD4 and CD8 T cells in immune organs when compared to groups receiving no vaccine and blank microneedles (*P ⁇ 0.05).
  • splenocytes from the various groups were plated onto a 48 well plate and re- stimulated with antigen for 16 hours and then stained with fluorescently tagged antibodies for CD4 and CD8 cells and quantified using flow cytometry (Fig. 11).
  • the groups receiving the vaccine via the subcutaneous or transdermal route showed significantly higher cell counts than the groups which did not receive the vaccine (*P ⁇ 0.05).
  • breast cancer affects one in eight women during their lives. It kills more women in USA than any other cancer.
  • Current treatment strategies act non-specifically against both tumor cells and normal cells.
  • the immunized animals showed significantly lower tumor growth compared to the naive animals that did not receive any treatment.
  • Breast cancer is the most commonly diagnosed malignancy and is the second leading cause of cancer related death in American women. This year over 250,000 women will be diagnosed with breast cancer and almost 50,000 women will die from metastatic breast cancer. Currently there are no FDA approved vaccines for breast cancer. For this reason development of a therapeutic breast cancer vaccine is an area of research that needs urgent attention offering these women a better chance of a cancer free life. Many therapeutic vaccine strategies are under clinical trials for breast and other types of cancers (1). Most vaccines being studied today, such as the gene transfer based vaccines require live cultured cells, which is time consuming and difficult to establish in many cancers. It is well known that breast cancer cells do not grow easily in vitro, significantly limiting the number of patients eligible for such clinical trials and ultimately vaccine therapy.
  • microparticles provide a depot from which the antigens a slowly release and cause a long lasting immune response. These microparticles protect the antigen from being cleared out from the body thus, enhancing the vaccine stability. This will be a major advantage from the standpoint of advancing the vaccine formulation from bench to clinic as scale-up of the process can easily be achieved with no further modifications.
  • MF59 have been incorporated in vaccine formulation to enhance the specific immune response generated by the antigen.
  • MF59 can potentiate the immune response by either increasing the antibody response and inducing cell mediated immunity i.e. they have a balanced strong Thl / Th2 stimulation.
  • the use of adjuvants not only enhances immunogenicity but could also permits the reduction in the antigen dose to be delivered in vaccine thus sparing the antigen.
  • MF59 is a squalene in water emulsion which is commercially been approved in Europe with more than 27 million doses of vaccine containing MF59 have been administered.
  • Novartis Vaccines has developed an influenza vaccine using MF59 along with inactivated, subunit seasonal prophylactic vaccine that is commercialized successfully as Fluad® in Europe.
  • the safety and efficacy of MF59 has been established clinically with a large database.
  • Treg can be suppressed before vaccination.
  • the dose of cyclophosphamide is much lower than the chemotherapeutic dose, thus there are no potential side effects to the patients.
  • the Treg cells remain depleted for a short period of 45 days and then again reach normal levels. Thus during the vaccination regimen the Treg levels are low and aid in generation of a strong immune response
  • the skin provides a unique site for the vaccination purposes as it is easily accessible and houses various immune cells for an efficient immune response against a range of antigens.
  • Skin serves as a barrier against various pathogens and is equipped with the skin associated lymphoid tissues (SALT) to combat any insult from invading pathogens.
  • SALT skin associated lymphoid tissues
  • Various skin cells assist in generation of effective immune response. Keratinocytes are the most pre-dominant (95%) epidermal cells in the skin. They can be activated by pathogens and result in production of cytokines, which in turn recruits dendritic cells/antigen-presenting cells to the site of action leading to initiation of the immune response.
  • Langerhans cells comprise of only 2% of the total cell population in the epidermis but due to their extended dendrites spread in the epidermal layer they cover over 25% of the skin surface. These are professional phagocytic cells efficient in immune surveillance and further signaling to the T-cells present in their vicinity. Activated macrophages and T-cells drain into nearby lymph nodes leading to an enhanced immune response.
  • Most of the vaccines are administered via subcutaneous or intramuscular route. These have been highly effective in generating protective immune response but they remain to be invasive, painful and require a skilled professional for vaccination. In an attempt to minimize some of these issues scientists have explored the potential of delivering particulate based vaccine antigens intradermally using microneedles formulated in our laboratory as described earlier.
  • Microneedle arrays are micron-sized needles, which upon insertion into the skin result in formation of aqueous conduits forming a passage for the vaccine antigens towards the immune-competent skin layers. Due to their short needle length, they avoid contact with the nerve endings in the dermis thus remain to be a painless mode of immunization. This opens a new avenue of vaccine delivery through an effective, painless and patient-friendly route of administration. The success of immunization via skin using microneedles inspired us to evaluate the potential of delivering a breast cancer vaccine through this route. This approach further can potentially be translated to a clinical setting where the patient undergoes a surgery for removal of the tumor and these tumor cells can serve as source of antigens for an individualized particulate vaccine, which can be administered therapeutically to avoid relapse
  • microparticulate vaccine formulation for metastatic breast cancer by using a murine metastatic breast cancer cell line 4T1 for transdermal administration through microneedles.
  • the microneedle based microparticulate vaccine formulation is a viable dosage form that may result in significant reduction in tumor growth as well as prevention of metastasis in murine model.
  • Aim 1 To prepare, characterize and evaluate the immunogenicity of biodegradable 4T1 metastatic breast cancer vaccine microparticles
  • Aim 2 To evaluate the cell surface expression on dendritic cells treated with the microparticulate vaccine formulation.
  • Aim 3 To determine the efficacy of the particulate 4T1 metastatic breast cancer vaccine administered by the microneedle based transdermal route in murine breast cancer model
  • whole cell lysate for murine breast cancer cell line (4T1) was prepared by using hypotonic lysis buffer (10 mM Tris and 10 mM NaCl) and further subjected to five freeze thaw cycles at -80°C and 37°C for 10 minutes each. At the end of last freeze thaw cycle, cell lysis was confirmed using trypan blue dye exclusion assay; presence of dead cells confirmed the end point.
  • the whole cell lysate (WCL) thus obtained was stored at -80°C for further use.
  • the 4T1 antigen loaded vaccine particles were prepared by spray-drying an aqueous suspension containing whole cell lysate WCL, ethyl cellulose, cellulose acetate phthalate (CPD), hydroxyl-propyl methylcellulose acetate succinate (HPMCAS) and trehalose using the following formula :
  • Whole cell lysate WCL 10% w/w
  • Ethyl cellulose 35% w/w
  • Cellulose acetate phthalate (CPD) 25 % w/w
  • Hydroxyl-propyl methylcellulose acetate succinate (HPMCAS) 25 %
  • Trehalose 5% w/w.
  • Adjuvant microparticles were prepared following the same procedure with adjuvant loading of 2.5% w/w.
  • Vaccine and adjuvant microparticles were incorporated into dissolving microneedle patches for vaccine administration.
  • PDMS polydimethylsiloxane
  • Spray dried particles were analyzed for their size and zeta potential.
  • Antigen loaded microparticles were suspended in citrate buffer (100 mM, pH 4.0) and particle size was measured using Spectrex laser particle counter (Spectrex Corp. CA). Zeta potential of these microparticles was measured using Malvern Zetasizer Nano ZS (Malvern Instruments, Worcs, UK).
  • vaccine microparticles were visualized using scanning electron microscope (Phenom World Pure Scanning electron microscope). The particle size was within a range of 1-4 ⁇ . The size and shape of particles was confirmed using scanning electron microscopic images (Figs. 12A and 12B).
  • the particles are doughnut shaped and porous in nature.
  • the particles have a positive zeta potential of 7 to 9 mV. Positive zeta potential helps prevent aggregation and aids uptake of particles by dendritic cells.
  • Nitric oxide assay is an important marker for innate response. Antigen-presenting cells like dendritic cells release nitric oxide upon exposure to an antigen. In this study we found that there is significantly higher amount of nitric oxide released in the supernatant of cells exposed to vaccine microparticles compared to vaccine solution and blank microparticles. Vaccine microparticles induced nitric oxide release of 70.03+ 10.32 ⁇ of nitrite compared to 10.37+ 4.21 ⁇ of nitrite by lysate solution (see Fig. 13).
  • DCs Dendritic cells
  • Fig. 15 is a graph of tumor volume measurement.
  • Figs. 16A-D are graphs of In vivo CD4 and CD8 T cell response in different treatment groups as percent cell count measurement of lymph nodes CD 4 T cells (Fig. 16A), spleen CD 4 T cells (Fig. 16B), spleen CD 8 T cells (Fig. 16C) and spleen CD 8 T cells (Fig. 16D).
  • Table 2 shows a progression of metastasis to lung, lymph node and liver in different treatment groups.
  • Bozeman EN Shashidharamurthy R, Paulos SA, Palaniappan R, D'Souza M, Selvaraj P. Cancer vaccine development: designing tumor cells for greater immunogenicity. Front Biosci Landmark Ed. 2010;15:309-20.
  • the Influenza virus is an RNA virus, part of the Orthomyxoviridae family. 1 There are three main subtypes of influenza: A, B and C. 1 Subtype A infects nonhuman hosts, however transmission from these hosts to humans can occur which can have a serious impact and lead to a pandemic crisis. 1 Subtypes B and C only infect humans. 1
  • Influenza is enclosed in a lipid bilayer that houses the RNA that codes for all the virus' proteins (Fig. 17).1
  • RNA that codes for all the virus' proteins
  • HA hemagglutinin
  • NA neuraminidase
  • M2 matrix 2 protein
  • the major goal of vaccines is to provide immunity, which is a term used to describe the ability of the host to protect itself against a foreign pathogen.4 This is dependent on the coordination of two major systems the innate and adaptive.4 Microorganisms such as bacteria and viruses that infiltrate the host for the first time are met by the innate immune system, which include macrophages that are activated and release proteins known as cytokines and chemokines that signal other cells to the site of infiltration.5 A vital class of immune cells that arrive to the site are dendritic cells (DCs) which help by engulfing these pathogens thus causing induction of the adaptive immune response.5 Following encounter with the pathogen, dendritic cells enter a developmental program referred to as maturation.6 The function of mature DCs is not to destroy pathogens, but to become highly effective antigen-presenting cells (APCs) and carry antigen
  • T lymphocytes There are three major subsets of T lymphocytes: cytotoxic, suppression and helper.4 Cytotoxic T lymphocytes aid in the destruction of cells that may be infected and recruit other cells to destroy the pathogen that caused the infection in these cells.4 Suppressor T lymphocytes coordinate the immune response by regulating the level of the response.4 Helper T lymphocytes activate other immune cells which aid in destruction of the pathogen, i.e. plasma cells to release antibodies.4 Vaccines are known to activate these machineries by first exposure of the antigen to immune cells, leading to recognition of the antigen following second exposure and
  • VLPs Viruslike particles
  • HPV human papillomavirus
  • vaccines In order to generate a dynamic immune response, vaccines should meet two main criteria, it should produce long-term protection and should be specific to the antigen.8 To meet these criteria, compounds known as adjuvants are used to enhance activation of the immune system.8 In the 1920's aluminum based compounds demonstrated excellent adjuvant activity and have been licensed and are widely used in human vaccines today.8 Research has shown that aluminum based adjuvants enhance immunogenicity by producing a depository at the site of administration allowing for sustained release of the antigen.8 This sustained release mechanism allows for increased interface between the antigen and cells of the immune system.8 One of the most common entry sites for infectious diseases including influenza is through mucosal membranes, therefore researchers have sought out the use of adjuvants that target mucosal membranes to improve vaccines.9 Immune cells express a family of proteins categorized as tolllike receptors which help in the elimination of pathogens, therefore adjuvants that target these toll-like receptors can enhance the immunogenicity of vaccines.10 Toll-like receptor 4
  • Influenza is one of the most devastating infectious diseases due to the ease of spread. Memory response after primary infection is critical, however due to the high rate of mutation of the virus; the antibodies produced in primary infection are not specific and are unable to protect against secondary influenza infection.
  • the immune system is composed of two major classes of immunity, innate and adaptive immunity. Adaptive immunity is subdivided into humoral and cell-mediated responses. The fight against influenza involves both humoral and cell-mediated immune responses. In addition to humoral mediated antibody production, activation of T lymphocytes, both CD4+ and CD8+ T cells are important for recovery against influenza infection. Priming of the host defense using vaccines is key for prevention of influenza infection.
  • Transdermal delivery of particulate vaccines has been shown to induce protective immunity against influenza, however the mechanism of this response is not well understood. It is thought that differentiation of T cells into Thl and Th2 cells is vital for delineation of immune cells that lead to immunological memory. Thl differentiation leads to CD8+ activation of cytokines that then help to convert to memory T cells. Th2 differentiation leads to CD4+ activation of cytokines that can lead to both T and B cell memory. In order to investigate this mechanism, the Thl and Th2 immune responses were explored following transdermal vaccination with a particulate system incorporating the A/PR/8/34 (HlNl) inactivated influenza virus and delivery using a micro needle injector.
  • HlNl A/PR/8/34
  • the particulate formulation was first prepared using the spray drying method.
  • the inactivated influenza virus was incorporated into a polymer matrix with the following composition: 20% (w/w) cellulose acetate phthalate (Aquacoat ® CPD), 35 % (w/w) HPMCAS and 33% (w/w) of ethyl cellulose (Aquacoat ® ECD).
  • Other constituents include trehalose (5%), chitosan (5%), tween 20(0.5%) and lastly Inactivated influenza virus (1.5%).
  • the microparticulates were visualized using transmission electron microscopy (Fig. 19). Size and charge are illustrated in Table 3.
  • Dissolving microneedles intended for the painless transdermal release of encapsulated pharmaceutical agents after dermal insertion, were developed as a solution to the safety issue.
  • Dissolvable microneedles mainly deploy PDMS micromolds which are made from a master structure of microneedles (Fig. 19). Briefly, Polydimethylsiloxane (PDMS) was poured onto the stainless steel master structure obtained from Micropoint Technologies INC, Singapore (Step 1-3; Fig. 19). The M2e VLP formulation was then spray dried respectively.
  • PDMS Polydimethylsiloxane
  • the microneedle formulation includes 10% of M2e VLP, 25% trehalose, 25% maltose, 20% polyvinyl alcohol and 20% hydroxypropylmethylcellulose acetate succinate (HPMCAS).
  • the formulation was prepared beginning with the addition of PVA, HPMC, Maltose and Trehalose to a microcentrifuge tube, followed by the addition of ammonium hydroxide (NH40H) to the microcentrifuge tube. Once dissolved, this formulation was then added to a microneedle mold avoiding air bubbles (Fig. 19). These molds were then placed straight into 50 mL centrifuge tubes. Centrifugation was done in a fixed angle centrifuge in order to remove air bubbles and to force the formulation into the microneedles mold.
  • NH40H ammonium hydroxide
  • the maximum speed was 2000 rpm which was achieved step wise, in order to avoid jerking in the rotation process, time for which centrifugation should be done was 5-10 min. After this centrifugation step, the backing layer was added and centrifuged repeatedly in the same manner. The molds were placed in in tubes and then in an incubator at 37 degrees C overnight.
  • Dendritic cells play a major role in immune system activation and are well known for their ability to release chemical messengers known as cytokines or chemokines, followed by activation of the adaptive immune system. Dendritic cells take up antigens and present them on major histocompatibility complexes I and II (MHC I, MHC II).
  • MHC I, MHC II major histocompatibility complexes I and II
  • the soluble and particulate vaccines (with and without adjuvants) were introduced to dendritic cells with the same amount of antigen and adjuvant.
  • the induced cells were incubated for 20 hours at 37oC with 5% C02.
  • Different markers can be used to evaluate the antigen presentation on APCs in ex-vivo or in-vitro studies. Some of these markers include CD40, MHC class II, CD80, and CD86. Both expressions of CD40 and MHC II on the surface of APCs are necessary for a successful stimulation of T cells from APCs as demonstrated in Figs. 20 and 21 (18).
  • CD80 and CD86 molecules are normally expressed on activated APCs and provide critical co- stimulatory signals for T cell activation and survival (19).
  • M2p MP group showed statistically higher CD 40 expression compared to M2p solution group. Except for alum, MF59, CpG and flagellin groups, all other adjuvant treated groups showed significant increase in CD40 expression levels compared to M2P MP alone.
  • Fig. 21 shows that the M2p MP group showed statistically higher CD 40 expression compared to M2p solution group. Except for P4 and Flag, MHC II expression was elevated in all adjuvant treated groups compared to M2p MP. Elevated MHC II expression will elicit a T-helper cell mediated response leading to antibody production against the antigen.
  • the microparticulate vaccine enhanced the CD80 expression but not the CD86 expression, compared to these expressions of the soluble vaccine group.
  • the M2p MP group showed statistically higher CD80 expression level compared to the M2p solution group.
  • CD 80 expression was elevated in Alum, CT, MF50, MPL, R848 and treated groups.
  • Fig. 23 there was no significant difference in CD 86 expression between soluble and microparticulate M2p vaccines.
  • CD 86 expression was elevated in R848, P4 and CpG treated groups compared to M2p MP.
  • Table 4 shows the group, treatment, route, dose and number of doses.
  • mice were challenged with a lethal dose of mouse adapted A/Philippines/2/82 (H3N2) live influenza virus. Mice were monitored daily to record changes in weight and mortality. Following 25% loss in body weight, animals were sacrificed for further analysis, by Mercer University IACUC review board and conducted under the guidelines of Mercer University IACUC.
  • lymph node was extracted (i.e. spleen and lymph node) and made into single cell suspensions.
  • the single cell suspensions were stained with fluorescence-conjugated antibodies specific to T cell phenotypes, helper T cells (CD4+) and cytotoxic T cells (CD8+) and quantified using flow cytometry. See Fig. 25.
  • Thery C Amigorena S. The cell biology of antigen presentation in dendritic cells. Curr Opin Immunol. 2001;13(1):45-51.
  • HPVs Human papillomaviruses
  • HPVs Human papillomaviruses
  • HPV serotypes (16 and 18) are responsible for approximately 70% of cervical cancers and precancerous cervical lesions (Centers for Disease Control and Prevention, 2015, p. 1).
  • Several symptoms of cervical cancer tend to appear only after the cancer has reached an advanced stage, which include irregular, intermenstrual or abnormal vaginal bleeding after sexual intercourse; back, leg or pelvic pain; fatigue, weight loss and loss of appetite. It takes 15 to 20 years for cervical cancer to develop in women with a normal immune system, while 5 to 10 years in women with a weakened immune system, such as those with untreated HPV infection.
  • GlaxoSmithKline GlaxoSmithKline are widely available in North America and Europe.
  • the Centers of Disease Control and Prevention (CDC) recommends boys and girls to get vaccinated against HPV, especially between ages of 9 to 26.
  • Both vaccines consist of HPV16 and HPV18 to prevent cervical cancers.
  • Gardasil® contained 9 different HPV serotypes (HPV 6, 11, 16,
  • vaccine solutions can be converted into micro/nano particles using a spray-drying or lyophilization process (Prathap Nagaraja Shastri et al., 2015).
  • spray drying process first evolved several decades ago, with the sudden need to reduce the transport weight of food and other materials (R. P. Patel, M.P. Patel, & A.M. Suthar, 2009).
  • spray drying process is extensively employed in the pharmaceutical field because of several advantages: (i) single step processing, (ii) easy to scale-up, and (iii) continuous processing operation.
  • the functional principle of the spray drying process is based on the atomization of a liquid feed into very small droplets within a hot drying gas leading to flash drying of the droplets into solid particles (Ano et al., 2011). The particles are then separated from the drying gas, using a cyclone and/or a filter bag, which yields a final spray dried product (Filipe Gaspar, 2014).
  • Vaccine nano/microparticles consist of a single or multiple antigens incorporated into a polymer matrix along with targeting ligands, adjuvants, and cytokines (shown as Fig. 26).
  • Antigens can be inactivated whole cells (virus, bacteria), a part of the cells (capsid proteins, DNAs, peptides, polysaccharides) or toxoids.
  • virus-like particles are made of capsid protein LI and L2 of HPVs and are used as antigens in Gardasil® and Cervarix.
  • Viruslike-particles are genetically engineered particles similar in size and structure to the virus but do not possess viral genomes, hence they lack the ability to replicate and cause infection in the host.
  • Polymer matrices such as poly(lactic-co-glycolic acid) (PLGA), hydroxypropyl methylcellulose (HPMC), hypromellose acetate succinate (HPMCAS), bovine serum albumin (BSA), cellulose acetate phthalate (CPD) and cyclodextrins (CDs) have been investigated as potential polymers. Selected polymers should be biodegradable and biocompatible in humans, to prevent any toxicity concerns.
  • Encapsulated antigens are portrayed as pathogens or foreign substances and are therefore taken up better by antigen presenting cells (APCs) and activate the innate and adaptive immune system.
  • APCs antigen presenting cells
  • Micro/nanoparticulate delivery systems that contain the antigen within a polymeric matrix, aid in the delivery of antigen/adjuvant for a prolonged period of time.
  • antigens delivered by a particulate carrier can enhance uptake by immune cells owing to their size, surface charge and morphology.
  • Adjuvants have been studied and employed in vaccines for decades in order to improve, expedite, and prolong specific immune responses produced by vaccine antigens including increase in antibody responses, induction of cell mediated immunity, and reduction in dose of antigen and the number of doses required for vaccination (13).
  • vaccine antigens including increase in antibody responses, induction of cell mediated immunity, and reduction in dose of antigen and the number of doses required for vaccination (13).
  • APCs antigen-presenting cells
  • c immune system modulation
  • Various adjuvants utilize their distinct effects via different mechanisms to stimulate the immune system, and hence it is essential to appoint appropriate adjuvants for a specific given antigen.
  • Adjuvants can be classified into two types: delivery system and immune potentiator (16). Some adjuvants function as antigen delivery systems such as alum, calcium phosphate tyrosine liposomes, virosomes, emulsions micro/nano particles (MF59, ISCOMS), and virus-like particles, because these particulate adjuvants increase antigen stability and allow them to be presented for an extended period of time (prolonging the signal of the antigen) (17). Delivery system based adjuvants are often taken up by phagocytosis into antigen presenting cells (APCs), and they can also induce an immune response, signaling and indirectly activating APCs.
  • APCs antigen presenting cells
  • Immune potentiators are purified components of bacterial cells or viruses; thus, they are recognized as “danger signals” by receptors present on immune cells (APCs) (15). Immune potentiators directly stimulate all the necessary signals for an immune response to an antigen.
  • a major category of immune potentiators is toll-like receptor (TLR) agonists, which activate signaling pathways to trigger innate immune responses.
  • TLR toll-like receptor
  • Some examples of adjuvants that act as TLR agonists include MPL and synthetic derivatives, muramyl dipeptide and derivatives, CpG oligonucleotides, alternative bacterial or viral components (flagellin), saponins, dsRNA, and resiquimod (16).
  • adjuvants elevate the amount of antigen that reach APCs and immune potentiators mainly activate these APCs; combinations of adjuvants from both classes can be used to maximize potency of a vaccine.
  • adjuvants along with the antigen which when used in combination enhance their ability to produce a robust immune response.
  • adjuvants when combined with the microparticulate vaccine will potentiate the immune response and result in improved efficacy by generation of an antigen- specific antibody response.
  • adjuvants enable the vaccine to produce long-term immunity in case of re- exposure to virus.
  • dendritic cells and Langerhans cells that reside in the dermal layer of the skin have the ability to capture the antigen, migrate to the secondary lymphoid organs and present the antigen to the T cells to generate adaptive immune responses (12).
  • transdermal delivery of the vaccine by encapsulating a viral antigen such as the HPV 16 VLP in a biodegradable matrix may not only result in better stability and uptake by Langerhans cells/ dendritic cells but will also provide enhanced antigen presentation and recognition by the immune system.
  • the innovation in our current approach lies in our proposed strategy, which is to develop, characterize and assess a micro/nanoparticulate vaccine against Influenza.
  • the goal of this study was to determine the formulation parameters of a microparticulate vaccine for Influenza using the HPV 16 VLP.
  • the HPV VLP was incorporated into an enteric-coated polymer matrix in this formulation.
  • This matrix consisted of celluloseacetatephthalate (CPD) hydroxypropylmethylcellulose acetate succinate (HPMCAS), ethylcellulose (EC), trehalose and glycol chitosan polymers
  • CPD dispersion (30% w/v) was diluted in deionized water with a concentration of five mg/ml under stirring.
  • CPD and HPMCAS were dissolved separately using IN sodium hydroxide to make final solutions at pH of 6.0 and 8.0, respectively.
  • the mixture of CPD, HPMCAS, and EC was obtained as mentioned above, and the final solution pH was adjusted to be 7.0. Glycol chitosan was then added along with HPV 16 VLP and trehalose. In addition, Tween-20 was added to enhance the smooth surface of the microparticles (MPs). The solution was stirred at 50 rpm during the spraying process using a Buchi B290 spray dryer to maintain its homogeneity. Microparticulate adjuvants were formulated using the same procedure as the vaccine microparticles.
  • microparticles See Fig. 27
  • SEM Phenom Pure Desktop® scanning electron microscopy
  • Fig. 28 is a graph of nitric oxide concentration of various solutions.
  • markers can be used to evaluate the antigen presentation on APCs in ex- vivo or in-vitro studies. Some of these markers include CD40, MHC class II, CD80, and CD86. Both expressions of CD40 and MHC II on the surface of APCs are necessary for a successful stimulation of T cells from APCs as demonstrated in Figs. 29 and 30 (18). In this study, the particulate vaccine enhanced both MHC II and CD40 expressions (p ⁇ 0.001), compared to the soluble vaccine. Except P4 and flagellin, all other adjuvants enhanced highly MHC II expression (p ⁇ 0.001) on the cell surface, compared to the M2p MP group.
  • CD80 and CD86 molecules are normally expressed on activated APCs and provide critical co-stimulatory signals for T cell activation and survival (19).
  • the microparticulate vaccine enhanced the CD80 expression but not the CD86 expression, compared to these expressions of the soluble vaccine group.
  • all other adjuvants induced statistically (p ⁇ 0.001) the CD80 expression.
  • R848 and CpG enhanced the highest (p ⁇ 0.001) expression of CD86, followed by alum and P4 treated groups, compared to the vaccine MP treated group.
  • HPV 16 VLP human papilloma virus type 16 virus-like particles
  • the HPV 16 VLP was incorporated into a cellulose polymer mix of cellulose acetate phthalate (Aquacoat® CPD), HPMCAS and ethyl cellulose (Aquacoat® ECD).
  • Other constituents included trehalose, chitosan, tween 20 and, lastly, the HPV 16 VLP. This suspension was spray dried using the B- 290 spray dryer to obtain particulates.
  • Dissolving microneedles intended for the painless transdermal release of encapsulated pharmaceutical agents after dermal insertion, were developed as a solution to the safety issue.
  • Dissolvable microneedles mainly deploy PDMS micromolds which are made from a master structure of microneedles as illustrated below. Briefly, Polydimethylsiloxane (PDMS) was poured onto the stainless steel master structure obtained from Micropoint Technologies INC, Singapore. The HPV 16 VLP formulation was then spray dried respectively. The microneedle formulation included 10% of M2e VLP, 25% trehalose, 25% maltose, 20% polyvinyl alcohol and 20% hydroxypropylmethylcellulose (HPMC).
  • PDMS Polydimethylsiloxane
  • the formulation was prepared beginning with the addition of PVA, HPMC, Maltose and Trehalose to a microcentrifuge tube, followed by the addition of Ammonium hydroxide (NH40H) to the microcentrifuge tube. Once dissolved, this formulation was then added to a microneedle mold avoiding air bubbles. These molds were then placed straight into 50 mL centrifuge tubes. Centrifugation was done in a fixed angle centrifuge in order to remove air bubbles and to force the formulation into the microneedles mold. The maximum speed was 2000 rpm, which was achieved step wise in order to avoid jerking in the rotation process, time for which centrifugation should be done was 5-10 min. After this centrifugation step, the backing layer was added and centrifuged repeatedly in the same manner. The molds were placed in tubes and then in an incubator at 37 degrees C overnight.
  • mice antibody responses were monitored for a 40-week period, followed by which they were sacrificed for further analysis.
  • the groups for the study are shown in Table 5.
  • Samples were stored in -20 °C until analysis. Specific serum antibody for HPV 16 was assessed using ELISA. Plates were coated with 100 ⁇ g/well of HPV VLPs. Serum samples were used for detection of primary antibody and alkaline phosphatase (AP) conjugated goat anti-mouse IgG was used as secondary antibody to determine total amount of antibody. The alkaline phosphatase substrate was used for detection of color. The optical density was taken at 405nm on the Biotek Synergy ELISA plate reader.
  • AP alkaline phosphatase
  • the primary and secondary lymphoid organs were extracted (i.e. spleen and lymph node) and made into single cell suspensions.
  • the single cell suspensions were stained with fluorescence conjugated antibodies specific to T cell phenotypes, helper T cells (CD4+) and cytotoxic T cells (CD8+) and memory B and T cell phenotypes, memory T cells (CD45R and CD62L) and memory B cell (CD27) quantified using flow cytometry.
  • Roper RL Antigen presentation assays to investigate uncharacterized immunoregulatory genes. Methods Mol Biol Clifton NJ. 2012;890:259-71.
  • Respiratory Syncytial Virus is the leading cause of bronchiolitis in infants and immunocompromised adults. It is estimated that approximately 3.4 million children are annually hospitalized due to RSV-related illnesses and 160,000 people die from RSV infection worldwide (Nair et al., 2010). The past few decades have been spent in developing a promising strategy to combat the virus either using subunit vaccines, attenuated viruses or live vector vaccines. With the centralized controversy surrounding the disease i.e., the tragic outcome of vaccinated children who developed vaccine-enhanced respiratory disease, in the 1960s with alum-adjuvanted, formalin inactivated RSV; there still remains a large barrier before the licensure of an RSV vaccine (H. W.
  • RSV has 10 genes that encode 11 proteins. Among them are the F, fusion protein and G, glycoprotein which are important antigenic proteins expressed on the surface of the virus and a target for neutralizing antibodies that facilitate a protective immune response in the patient (Murawski et al., 2010).
  • VLPs Virus-like-particles
  • Recombinant baculovirus-expressed VLPs containing RSV-F and/or G glycoproteins were shown to stimulate antigen- specific antibody responses and defend against RSV infection in murine models (K.-H. Kim et al., 2015; Murawski et al., 2010; Quan et al., 2011).
  • Encapsulated antigens are portrayed as pathogens or foreign substances and are therefore taken up better by antigen presenting cells (APCs) and activate the innate and adaptive immune system.
  • APCs antigen presenting cells
  • Micro/nanoparticulate delivery systems that contain the antigen within a polymeric matrix, aid in the delivery of antigen/adjuvant for a prolonged period of time.
  • Several publications from our lab have proved the efficacy of using microparticles for delivering cancer and infectious disease antigens through the oral, transdermal and subcutaneous routes.
  • LCs Langerhans cells
  • Adjuvants are crucial compounds used in combination with vaccine antigens to enhance their ability to produce a stronger immune response. They are minimally toxic and have no long lasting immune effects when given alone. Specific aims 1 and 2 have used adjuvants with vaccine to potentiate the immune response against the disease.
  • Alum an adjuvant delivery system has been widely used in human vaccines for decades. The hypothesized mechanisms of Alum include enhanced antigen uptake by APCs, improved MHC II expression and antigen presentation (Dubensky & Reed, 2010).
  • Monophosphoryl lipid A (MPL®) is a Toll-like receptor-4 agonist that induces a strong cellular (T cell mediated) immune response.
  • MF59TM Another approved adjuvant, MF59TM is a squalene in water nano-emulsion that shows cell-mediated/ antibody responses and results in secretion of cytokines and chemokines by DCs and macrophages.
  • Pneumococcal surface adhesion A-derived peptide (P4) has been recently explored as an adjuvant since it has shown enhanced opsonophagocytosis in some studies (Rajam, Anderton, Carlone, Sampson, & Ades, 2008).
  • R848 is an imidazoquinoline compound that activates immune cells via the TLR7/8 pathway.
  • VLPs F Fusion protein virus-like particles
  • a unique blend of cellulose polymers and chitosan was utilized to formulate microparticles. Briefly 0.5 % (w/w) F-VLP solution was incorporated into a mixture of cellulose acetate phthalate, hydroxypropylmethylcellulose acetate succinate (HPMCAS), ethylcellulose, trehalose and chitosan polymers. The aqueous suspension was subsequently spray-dried using the Buchi-290 spray dryer to obtain micro/nanoparticles. The microparticles were characterized and analyzed for their size, surface charge, encapsulation efficiency and antigen stability.
  • HPMCAS hydroxypropylmethylcellulose acetate succinate
  • the Malvern Zetasizer® Nano ZS was used to carry out size and surface potential measurements. Particles were suspended in citric acid (lOmM, pH 3.8) for 10 minutes and centrifuged. The particles were resuspended in deionized water and later analyzed by the instrument. The microparticle images were captured on carbon sheets and observed under 20kV at 7500X using the scanning electron microscope (See Fig. 34). [00271] Table 6 summarizes the physicochemical characteristics of the spray dried microparticles.
  • Dendritic cells are responsible for pathogen recognition and eradication by releasing cytokines such as TNF-a, nitric oxide (NO) and IFN- ⁇ .
  • cytokines such as TNF-a, nitric oxide (NO) and IFN- ⁇ .
  • NO nitric oxide
  • IFN- ⁇ IFN- ⁇
  • the increase in levels of nitrite may be related to enhanced antigen recognition and delivery to dendritic cells.
  • NO is also known to eradicate viruses by nitrosation of cysteine residues within key proteins required for replication purposes (Wink et al., 2011).
  • the microparticles' ability to generate an innate immune response was evaluated using the nitric oxide assay.
  • Cells were plated in 24 well plates following which blank MPs, RSV F-VLP suspension and vaccine MPs were added and incubated with the cells for 20 hrs.
  • the VLP-MPs were compared with controls of blank MPs and VLP solution.
  • the cell supernatant was analyzed for nitric oxide (NO) levels (Ubale, D'Souza, Infield, McCarty, & Switzerland, 2013) using the Griess Test.
  • NO nitric oxide
  • FIG. 36 shows the amount of nitric oxide released ( ⁇ ) from DC 2.4 cells when exposed to Cells Only, Blank MP, RSV F-VLP Suspension, RSV F-VLP MP (*p ⁇ 0.05). There was a significant release of nitric oxide seen in supernatant of cells receiving RSV F-VLP MPs.
  • Fig. 37 shows Amount of nitric oxide released from DC 2.4 cells when exposed to VLP Suspension, RSV VLP MP and RSV VLP MP + Alum, MPL A and MF59 (*p ⁇ 0.05) There was a significant release of nitric oxide seen in supernatant of cells receiving RSV F-VLP + Alum/ MF59 MPs.
  • the cells were washed with PBS and detached using Trypsin EDTA. Each group of cells was analyzed separately for different markers (CD40, CD80, CD86, MHCI and MHC II) using the flow cytometer. Theoretically, dendritic cells will engulf antigen and adjuvant and process it in the phagolysosome. The protein fragments will be expressed on MHC II surface molecules. For the activation of the CD4+ T cell, a co- stimulatory molecule known as CD40 is required. CD40 and MHC II expression were significantly higher in the VLP-MP + Alum group compared to VLP solution. The inclusion of adjuvant increased CD40 expression and antigen presentation on MHCII molecules. Fig.
  • FIG. 38 is a graph showing the expression of CD40 on DC 2.4 cells exposed to Blank MP, F-VLP solution, F-VLP MP and VLP MP with adjuvant. MHC II expression was significantly higher in MP group compared to VLP solution. CD40 expression was higher in V+Alum group, compared to F-VLP MP group (*p ⁇ 0.05). Adjuvants resulted in higher CD40 costimulatory expression.
  • Fig. 39 is a graph showing the expression of CD80 on DC 2.4 cells exposed to Blank MPs, F-VLP solution, F-VLP MP and VLP MP with adjuvants. CD80 is a co- stimulatory molecule required for activation of CD8 T cells.
  • CD80 expression was significantly higher in V MP+Alum (**p ⁇ 0.01) /MPL A (*p ⁇ 0.05) group compared to RSV F-VLP MP alone. MF59 did not result in enhanced expression of CD80 molecules.
  • Fig. 40 is a graph showing the expression of CD40 on DC 2.4 cells exposed to Blank MP, F-VLP solution, F-VLP MP and VLP MP with adjuvant.
  • MHC II is a protein that expresses fragments of antigen to T cells of the immune system. MHC II expression was significantly higher in MP group compared to VLP solution.
  • CD40 expression was higher in V+ Alum group, compared to RSV F-VLP MP group (**p ⁇ 0.01). Adjuvants resulted in higher CD40 costimulatory expression.
  • the particles collected were weighed and samples were characterized for size, surface charge and morphology.
  • the Malvern Zetasizer® Nano ZS was used to carry out size and surface potential measurements. Particles were suspended in citric acid for 10 minutes and centrifuged. The particles were re-suspended in deionized water and later analyzed by the instrument. The microparticle images were captured on carbon tape and observed under 20kV at 7500X using the Phenom ® Desktop SEM. The spray-dried microparticles were added to PBS, vortexed for 10 minutes followed by incubation at 37°C to extract the protein antigens from the matrix.
  • the sample was centrifuged and supernatant was subjected to the micro BCA protein assay to quantify the amount of protein incorporated in microparticles.
  • the vaccine MPs demonstrated immunogenic properties when compared with its solution counterpart, as seen in Fig. 37.
  • the vaccine MPs + adjuvants were incubated with dendritic cells and incubated for 20 hrs.
  • the cell supernatant was analyzed for nitric oxide followed by CD40, CD80, CD86, MHC I and CD54 expression which was examined using the flow cytometer.
  • the adjuvants significantly increased nitric oxide and surface marker expression of MHC I and CD80 as seen in Figs. 37-39. Hence, adjuvants may be effective in potentiating the immune response.
  • Dissolving microneedles intended for the painless transdermal release of encapsulated pharmaceutical agents after dermal insertion, were developed as a solution to the safety issue.
  • Dissolvable microneedles mainly deploy PDMS micromolds which are made from a master structure of microneedles (Fig. 5). Briefly, Polydimethylsiloxane (PDMS) was poured onto the stainless steel master structure obtained from Micropoint Technologies INC, Singapore (Step 1-3; Fig. 5).
  • PDMS Polydimethylsiloxane
  • microneedles were made using 10% w/w microparticles, 25 % w/w trehalose, 25 % w/w maltose, 20 % w/w polyvinylalcohol (PVA), 20 % w/w hydroxypropylmethyl cellulose (HPMC). PVA, HPMC, Maltose and Trehalose to a 1.7 mL microcentrifuge tube.
  • the contents were dissolved in minimum possible amount of water (e.g. 200 mg of total solid content can be dissolved in 600 uL water) and vortexed.
  • the maximum speed is 2000 rpm which is achieved step wise, in order to avoid jerk in the rotation process, time for which centrifugation should be done is 5-10 min. Speed should be lowered gradually for same reason as above.
  • Step 3-6; Fig. 5 This step can be repeated further by adding more formulation or blank backing layer solution.
  • the molds were placed in tubes and placed in an incubator at 37°C overnight (Step 7-9; Fig. 5).
  • mice were immunized with 1 prime and 2 booster doses of microparticulate RSV F-VLP via the transdermal route by treating the mice using dissolving microneedles.
  • Blood samples were collected every week and serum antibody (IgG) levels were analyzed by ELISA.
  • the mice were challenged with live RSV-A2 virus and body weight was measured for a period of 5 days.
  • effector T cell populations specifically, CD4+ and CD8+ T cells were quantified in the lymph node and spleen using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the lungs were harvested and utilized for histopathology staining and lung viral titer experiments. The lung viral load in the test groups helped us understand whether the vaccination protocol was effective in generating an immune response against the RSV infection.
  • Fig. 41 is a timeline for the animal study, with dosing and sampling intervals incorporated.
  • Fig. 42 is a graph showing IgG antibody levels in blood serum of mice inoculated with Inactivated RSV vaccine (FI-RSV), solution form of F-VLP, F-VLP microparticles and F- VLP + MPL microparticles.
  • Fig. 43 is a graph showing body weight measurements of mice 6 days post-challenge with live RSV A2 virus. Untreated mice (PBS) showed the highest change in weight compared to vaccinated mice.
  • Fig. 44 and Fig. 45 show graphs of CD4+ and CD8+ T cell response after challenge with live RSV A2 virus.
  • TD MP+MPL showed higher CD4 and CD8 T cell populations compared to TD MP and TD Suspension + MPL.
  • FIG. 46 is a graph of viral titers measured in lung homogenates of various groups after challenge using RT-PCR. Lung viral titers were found to be significantly higher in mice vaccinated with inactivated RSV given IM, VLP suspension and VLP microparticles given transdermally compared with VLP MP + MPL A.
  • Virus-like particle vaccine containing the F protein of respiratory syncytial virus confers protection without pulmonary disease by modulating specific subsets of dendritic cells and effector T cells. Journal of Virology, JVI.02018-15. http://doi.org/10.1128/JVI.02018-15
  • Diabetes mellitus is a chronic metabolic disease and is one of the primary causes of mortality in well developed countries.
  • the causative factor responsible for none or underproduction of insulin is due to destruction of pancreatic beta cells in type I diabetic patients.
  • the first line therapy is to inject insulin directly to patients or either organ transplant.
  • Our strategy is to enclose (encapsulate) pancreatic beta cells in polymeric microcapsules. This technology works by encapsulating the cells in a semipermeable membrane that allows the entry and exit of small molecules like oxygen and proteins like insulin (Mol wt - 6 KDa These cells can produce the protein of interest de novo and deliver the biotherapeutic molecules in the body.
  • the advantage of our proposed strategy over existing therapy would limit the dosing frequency and circumvent the need for organ transplantation
  • microcapsules were prepared by spraying a sucrose- alginate-beta cell suspension mixture into calcium chloride solution using a specialized spray nozzle. Calcium alginate microcapsules containing cells were coated with chitosan glutamate to form a semipermeable membrane at the surface. Various concentrations of sodium alginate, chitosan glutamate and calcium chloride were varied for optimal size and sphericity.
  • alginate solution (1.2%w/v) was prepared and beta islet cells were added to it and allowed to stir for fifteen minutes. After stirring, the alginate cell suspension was then sprayed via 1.40 mm nozzle using a Buchi spraying apparatus in calcium chloride solution (1.5%w/v). Microcapsules in calcium chloride suspension were allowed to stir for fifteen minutes and washed with PBS, centrifuged twice at x 285g (1200 rpm) to remove the excess calcium ions.
  • Alginate suspension was then transferred to chitosan glutamate (0.5%w/v) solution and stirred further for fifteen minutes and washed with PBS, centrifuged twice at x 285g (1200 rpm) to remove the excess chitosan and finally transferred in DMEM media and kept in the incubator at 37oC.
  • Different sized microcapsules can be achieved by spraying the alginate suspension at different gas flow rate. Maintaining the higher gas flow rate leads to reduction in size of microcapsules due to high shear at the tip of nozzle.
  • Microcapsule size was measured by using light optical microscope. The size distribution was evaluated at mean size of 300 ⁇ obtained at a gas flow rate of 250 L/hr. Size distribution was determined by taking a total of 50 microcapsules.
  • Fig. 49 is a graph of the size of microcapsules (diameter being in ⁇ ) plotted at different gas flow rates. Plotted values are mean with + standard deviation bars.
  • Fig. 50 is a graph of microcapsule size distribution obtained after spraying the alginate suspension at 250 L/Hr.
  • microcapsule size was found to be in the range of 200-400 ⁇ with a mean of 300 ⁇ .
  • FTIR analysis was performed to confirm the cross linking of alginate microcapsules with calcium chloride used during the fabrication process. Spectra was taken for powdered samples of sodium alginate and compared with the spectra obtained from dried microcapsules. Disappearance of peaks in the region of 3250 cm-1 in alginate microcapsules suggest crosslinking of matrix of alginate microcapsules by calcium chloride added during the fabrication of microcapsules as cross linking agent.
  • Fig. 52 is a graph of FTIR spectra of sodium alginate.
  • Fig. 53 is a graph of FTIR spectra of alginate microcapsules.
  • FIG. 54A is a light microscopic image showing clusters of microcapsules encapsulated beta islet pancreatic cells were taken at magnification of 10X.
  • Fig. 54B is a light microscopic image showing clusters of microcapsules encapsulated beta islet pancreatic cells were taken at magnification and 40X.
  • Cell viability in the microcapsules is required for the beta cells to secrete insulin in response to glucose concentration.
  • Cell viability was measured using fluorescent Live/Dead Staining kit.
  • the living cells are stained green by the fluorescent calcein that is hydrolyzed from non-fluorescent calcein AM by the intracellular esterases.
  • Ethidium homodimer-1 (EthD-1) enters only the damaged cells and yields increased red fluorescence signal upon binding to nucleic acids. Fluorescent images were taken at different time over a period of thirty days. Stained cells were observed under fluorescent microscope and photographed with a digital camera.
  • Fig. 55 is a chart of Live dead cell stained images of microcapsules encapsulated pancreatic islet beta cells collected over thirty days period at magnification of 10X.
  • Nitric oxide is an important marker for innate immune response. Antigen presenting cells like dendritic cells release nitric oxide upon exposure to an antigen. In this study we found that there is significantly higher amount of nitric oxide released in the supernatant of dendritic cells exposed to naked beta islet cells compared to microencapsulated cells.
  • Figs. 56A and 56B are graphs of nitric oxide release vs MC Blank (Microcapsules without beta islet pancreatic cells), MC cells (Microcapsules encapsulate beta islet pancreatic cells and Cells only (Unencapsulated cells) ns-not significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant.
  • Fig. 58 is a graph of long term stability monitored by measuring the fraction of intact microcapsules.
  • mice were segregated into different groups based on treatment given.
  • Group received microcapsules encapsulated pancreatic beta islet cells were injected intraperitoneally with microcapsules encapsulating beta cells equivalent to 3 million approximately. Unencapsulated cells group were injected with cells equivalent to 3 million cells.
  • Diabetic control group did not receive any treatment and healthy animals were used in the study for comparison with treatment groups.
  • blood glucose levels were measured after every 7 days and for a period of thirty five days. As shown in Fig. 59, it was found that the blood glucose levels were below 150mg/dL in group received microencapsulated beta cells.
  • Fig. 60 is a Kaplan Miers survival curve shows that the microcapsule group shows graft rejection on day 42.
  • Fig. 60 shows percent graft survival plotted for different groups i.e., Diabetic control, MC cells (Microcapsules encapsulated pancreatic beta islet cells) and Cells only (Unencapsulated cells).
  • Fig. 61 Measurement was taken at every 5 day interval and change in weight of mice was observed during the course of study. The data obtained suggest that the increase in weight was higher in groups administered with encapsulated cell in microcapsule. However the diabetic control group not received any treatment shows slight increase in weight.
  • mice in all groups sacrificed and immune organs i.e. spleen and lymph nodes were collected.
  • Single cell suspension was prepared by passing the spleen and lymph nodes cells through 40 ⁇ strainer. Spleen cells obtained were treated with RBC lysis buffer and centrifuged. This process continues till the cell suspension obtained was colorless. Then, cells of spleen and lymph nodes were seeded in 48 well plate and incubate with markers of CD4 and CD8 cells. After one hour of incubation, cells were washed to remove excess marker and were analyzed using flow cytometry.
  • Figs. 62A and 62B are graphs of percentage of CD4 and CD8 positive cells plotted for different groups i.e., Diabetic control, MC beta and naked Cells Only. *p ⁇ 0.05 significant, **p ⁇ 0.01 very significant, ***p ⁇ 0.001 extremely significant (spleen cells).
  • Fig. 62B does not show any statistical significance in expression of CD8 in cells only group and microcapsule group but shows scientific significance.
  • CD4 expression in lymph nodes cells follows the same trend as in spleen cells and it was higher in microcapsule cell group and it was statistically very significant (p ⁇ 0.01) in comparison to cells only group as shown in Figs. 63A and 63B (graphs of percentage of CD4 and CD8 positive cells plotted for different groups i.e., Diabetic control, MC beta and naked Cells Only. *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant (Lymph node cells).
  • CD8 expression in lymph nodes also follows similar trend as in spleen cells and it was higher in case of cells only group shows statistically extremely significant (p ⁇ 0.001) in comparison to microcapsule group and therefore confirms the protective ability of alginate microcapsule to beta islet cells in response to body immune response.
  • FIG. 64 is a graph of a flow cytometric analysis showing CD45R cell counts in different groups of mice *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant.
  • Fig. 65 is a graph of a flow cytometric analysis showing CD62L cell counts in different groups of mice *p ⁇ 0.05 significant, **p ⁇ 0.01very significant, ***p ⁇ 0.001 extremely significant. It was found that antibody secretion similar in all groups and no statistical difference was found.
  • CD62L a marker of naive T cells shows higher expression in cells only group and statistical very significant difference (p ⁇ 0.01) in comparison to microencapsulated cells group demonstrate less immune response developed for beta islet cells encapsulated in microcapsules.
  • CLAUSE 1 A transdermal delivery system, comprising: (a) at least one layer of a support material; (b) at least one biodegradable needle associated with the support material, each needle comprising i. at least one biodegradable polymer, ii. at least one sugar; wherein each biodegradable needle is adapted to retain a bioactive material.
  • CLAUSE 2 The system of Clause 1, wherein the biodegradable polymer is polydimethylsiloxane.
  • CLAUSE 3 The system of Clause 1, wherein the sugar is at least one material selected from the group consisting of maltose and trehalose.
  • CLAUSE 4 The system of Clause 1, wherein the bioactive material is at least one material selected from the group consisting of drugs, vaccines, and proteins, and mixtures thereof.
  • CLAUSE 5 The system of Clause 1, wherein the bioactive material is at least one material provided in microencapsulated particle form.
  • CLAUSE 6 The system of Clause 1, wherein the at least one biodegradable microneedle is able to degrade within 12 minutes after delivery to a subject's skin.
  • CLAUSE 7 The system of Clause 1, wherein the bioactive material is in the form of microencapsulated material having an average particle size in a range of 0.01-50 ⁇ .
  • CLAUSE 8 The system of Clause 1, wherein the bioactive material is in the form of microencapsulated material having an average particle size in a range of 1-4 ⁇ .
  • CLAUSE 9 The system of Clause 1, further comprising an adjuvant.
  • CLAUSE 10 The system of Clause 1, wherein the bioactive material is at least one material selected from the group consisting of a vaccine to gonorrhea, breast cancer, a vaccine to influenza, a vaccine to human papilloma virus, and a vaccine to respiratory syncytial virus.
  • CLAUSE 11 A method of forming a transdermal delivery system, comprising:
  • CLAUSE 13 The method of Clause 11, wherein the at least one biodegradable microneedle is essentially free of organic solvents.
  • CLAUSE 14 The method of Clause 11, wherein the polymer material comprises polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • CLAUSE 15 The method of Clause 11, wherein the sugar is selected from at least one sugar selected from the group consisting of trehalose, maltose, and mixtures thereof.
  • CLAUSE 16 The method of Clause 11, further comprising providing polyvinyl alcohol (PVA).
  • PVA polyvinyl alcohol
  • CLAUSE 17 The method of Clause 11, wherein the solvent of step (b) is essentially free of organic solvent material.
  • CLAUSE 18 A biodegradable microneedle, comprising: at least one biodegradable needle associated with the support material, each needle comprising at least one biodegradable polymer, and at least one sugar, wherein each biodegradable needle is adapted to retain a bioactive material.
  • CLAUSE 19 A method of transdermally delivering a bioactive material, comprising: (a) forming at least one biodegradable microneedle from at least one biodegradable polymer and at least one sugar; (b) associating a bioactive material with the at least one microneedle; (c) associating the at least one microneedle with a backing layer; and, (d) contacting the at least one microneedle containing the bioactive material with the skin of a subject; whereby the at least one microneedle introduces the bioactive material to the subject and the at least one microneedle biodegrades.
  • CLAUSE 20 A method of forming a transdermal delivery system, comprising:
  • step (a) mixing PVA, HPMC, and at least one sugar in a vessel; (b) dissolving the mixture of step (a) in water to form a mixture; (c) adding ammonium hydroxide to the mixture of step (b) and mixing; (d) adding to the mixture of step (c) at least one bioactive material in microencapsulated form to form a formulation; (e) adding an aliquot of the formulation of step (d) to a microneedle mold; and, (f) centrifuging the microneedle mold and formulation of step (e) to force the formulation into the microneedle mold.
  • CLAUSE 21 The method of Clause 20, further comprising step (g) associating a backing layer solution with the microneedles.
  • CLAUSE 22 The method of Clause 21, further comprising step (h) incubating the microneedle mold and backing layer to form a microneedle patch.
  • CLAUSE 23 The method of Clause 20, further comprising, after step (f), repeating steps (e) and (f) at least once.
  • Ranges may be expressed herein as from “about” one particular value, and/or to

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Abstract

La présente invention concerne un système d'administration transdermique à base de micro-aiguilles contenant un matériau bioactif, comprenant au moins une couche d'un matériau de support ; au moins une aiguille biodégradable associée au matériau de support, chaque aiguille comprenant au moins un polymère biodégradable et au moins un sucre, chaque aiguille biodégradable étant creuse et conçue pour retenir un matériau bioactif.
PCT/US2017/061353 2016-11-14 2017-11-13 Système d'administration transdermique à base de micro-aiguilles et son procédé de fabrication WO2018089918A1 (fr)

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US11666239B2 (en) 2017-03-14 2023-06-06 University Of Connecticut Biodegradable pressure sensor
US11745001B2 (en) 2020-03-10 2023-09-05 University Of Connecticut Therapeutic bandage
US11826495B2 (en) 2019-03-01 2023-11-28 University Of Connecticut Biodegradable piezoelectric ultrasonic transducer system

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Publication number Priority date Publication date Assignee Title
US11666239B2 (en) 2017-03-14 2023-06-06 University Of Connecticut Biodegradable pressure sensor
US11826495B2 (en) 2019-03-01 2023-11-28 University Of Connecticut Biodegradable piezoelectric ultrasonic transducer system
US11745001B2 (en) 2020-03-10 2023-09-05 University Of Connecticut Therapeutic bandage
WO2022063990A1 (fr) * 2020-09-28 2022-03-31 Dbv Technologies Particule comprenant une protéine rsv-f destinée à être utilisée dans la vaccination contre le rsv

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