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Poster Abstracts

2021 Poster Abstracts

Cell Therapy & Tissue Engineering

Exploring the potential of bioengineered cell-based therapy for Parkinson's disease


Priyal Bagwe (1,2); D. Joshi (1,2); N. Chitre (2); A. Bansal (1,2); K. Murnane (2,3,4); and MJ D'Souza (1,2) 1. Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University 2. Department of Pharmaceutical Sciences, Mercer University College of Pharmacy 3. Department of Pharmacology, Toxicology & Neuroscience, School of Graduate Studies, Louisiana State University Health Sciences Center 4. Department of Psychiatry, School of Medicine, Louisiana State University Health Sciences Center BACKGROUND Parkinson's disease (PD) is the second most common neurodegenerative disorder, associated with decreased dopamine levels in the brain. The current treatments for Parkinson's disease include symptomatic relief with drugs. The goal of this study was to assess the potential of a regenerative medicine-based cell therapy approach to increase dopamine levels. In this study, we used rat adrenal pheochromocytoma (PC12) cells that can produce, store, and secrete dopamine. These cells were microencapsulated in the selectively permeable polymer membrane to protect them from immune responses. METHODS PC12 cells were suspended in sodium alginate solution containing trehalose. The cell suspension was sprayed using the modified spraying device through 0.7 mm nozzle into calcium chloride solution. The microcapsules were then transferred to chitosan glutamate solution to coat the alginate membrane. The microcapsules obtained were then transferred to the culture media and stored in the incubator. Efficiency of the microencapsulation process was calculated as the percentage of viable cells after the encapsulation process using acridine orange dye. The parameters affecting the size of the microcapsules i.e., flow rate, pump speed, nozzle diameter, and concentration of sodium alginate were varied to optimize the size of microcapsules. FTIR-ATR spectra of sodium alginate and the microcapsules were recorded. The stability of the microcapsules was assessed. Immuno-isolation and selective permeability of the microcapsules were confirmed via micro-BCA protein assay and dopamine release assay. The release of nitric oxide by antigen-presenting cells was determined by Griess's assay. RESULTS Efficiency of the microencapsulation process was found to be 81.57 ± 4.3 %. The size of microcapsules reduced with increasing flow rate, increasing pump speed and by decreasing the nozzle diameter and concentration of sodium alginate. The air flow rate of 350 L/h, pump speed of 9 RPM, nozzle diameter of 0.7 mm, and 0.5 % w/v solution of sodium alginate produced microcapsules of an average size of 38.2 µm. FTIR-ATR spectra confirmed the crosslinking of sodium alginate with calcium chloride. PC12 cells were viable inside the microcapsules suspended in media for 30 days. Dopamine release assay showed that the microencapsulated cells could release a consistent amount of dopamine for 30 days. Micro BCA protein assay showed that BSA could not cross the microcapsule membrane. This confirmed the selective permeability of the microcapsule membrane. Griess's assay showed that the PC12 cells were significantly more immunogenic as compared to microcapsules encapsulating PC12 cells. CONCLUSIONS We could successfully formulate microcapsules encapsulating live PC12 cells. The microcapsules were found to be stable, and encapsulated cells were viable inside the microcapsules. The microcapsules were selectively permeable, thus could provide adequate protection to encapsulated cells, and were non-immunogenic. Taken together, PC12 microcapsules thus have the potential to serve as an alternative treatment approach for PD.





 
 

Drug Discovery & Development

Cell Therapy & Tissue Engineering

Exploring the potential of bioengineered cell-based therapy for Parkinson's disease


Priyal Bagwe (1,2); D. Joshi (1,2); N. Chitre (2); A. Bansal (1,2); K. Murnane (2,3,4); and MJ D'Souza (1,2) 1. Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University 2. Department of Pharmaceutical Sciences, Mercer University College of Pharmacy 3. Department of Pharmacology, Toxicology & Neuroscience, School of Graduate Studies, Louisiana State University Health Sciences Center 4. Department of Psychiatry, School of Medicine, Louisiana State University Health Sciences Center BACKGROUND Parkinson's disease (PD) is the second most common neurodegenerative disorder, associated with decreased dopamine levels in the brain. The current treatments for Parkinson's disease include symptomatic relief with drugs. The goal of this study was to assess the potential of a regenerative medicine-based cell therapy approach to increase dopamine levels. In this study, we used rat adrenal pheochromocytoma (PC12) cells that can produce, store, and secrete dopamine. These cells were microencapsulated in the selectively permeable polymer membrane to protect them from immune responses. METHODS PC12 cells were suspended in sodium alginate solution containing trehalose. The cell suspension was sprayed using the modified spraying device through 0.7 mm nozzle into calcium chloride solution. The microcapsules were then transferred to chitosan glutamate solution to coat the alginate membrane. The microcapsules obtained were then transferred to the culture media and stored in the incubator. Efficiency of the microencapsulation process was calculated as the percentage of viable cells after the encapsulation process using acridine orange dye. The parameters affecting the size of the microcapsules i.e., flow rate, pump speed, nozzle diameter, and concentration of sodium alginate were varied to optimize the size of microcapsules. FTIR-ATR spectra of sodium alginate and the microcapsules were recorded. The stability of the microcapsules was assessed. Immuno-isolation and selective permeability of the microcapsules were confirmed via micro-BCA protein assay and dopamine release assay. The release of nitric oxide by antigen-presenting cells was determined by Griess's assay. RESULTS Efficiency of the microencapsulation process was found to be 81.57 ± 4.3 %. The size of microcapsules reduced with increasing flow rate, increasing pump speed and by decreasing the nozzle diameter and concentration of sodium alginate. The air flow rate of 350 L/h, pump speed of 9 RPM, nozzle diameter of 0.7 mm, and 0.5 % w/v solution of sodium alginate produced microcapsules of an average size of 38.2 µm. FTIR-ATR spectra confirmed the crosslinking of sodium alginate with calcium chloride. PC12 cells were viable inside the microcapsules suspended in media for 30 days. Dopamine release assay showed that the microencapsulated cells could release a consistent amount of dopamine for 30 days. Micro BCA protein assay showed that BSA could not cross the microcapsule membrane. This confirmed the selective permeability of the microcapsule membrane. Griess's assay showed that the PC12 cells were significantly more immunogenic as compared to microcapsules encapsulating PC12 cells. CONCLUSIONS We could successfully formulate microcapsules encapsulating live PC12 cells. The microcapsules were found to be stable, and encapsulated cells were viable inside the microcapsules. The microcapsules were selectively permeable, thus could provide adequate protection to encapsulated cells, and were non-immunogenic. Taken together, PC12 microcapsules thus have the potential to serve as an alternative treatment approach for PD.





 

Food & Nutrition

A Biosensor for Rapid Detection of Listeria Monocytogenes for Food Safety Applications


Or Zolti(1*), Baviththira Suganthan(1*), and Ramaraja P. Ramasamy(1) 1. Nano Electrochemistry Laboratory, School of Chemical, Materials and Biomedical Engineering, University of Georgia (*Equal contribution) BACKGROUND Listeria Monocytogenes is a very common foodborne pathogen that has reported hospitalization rate of up to 90% of confirmed cases and death rate of up to 20% of confirmed cases according to the CDC and state health departments across the United States. These rates show the interest in developing an accurate, quick, and specific detection method to supplement or even replace conventional detection methods that are complex, time-consuming, requires trained personal, and are high cost. METHODS Electrochemical biosensors are good alternatives as diagnostic tools due to their ease of use, high specificity, and sensitivity. Bacteriophages can serve as excellent biorecognition elements in biosensors due to their ability discriminate between live and dead bacterial cells, their specificity, durability, and low cost. RESULTS An ultra-sensitive and highly selective electrochemical biosensor for detection of Listeria Monocytogenes has been designed, developed and tested in our laboratory. The sensor showed high selectivity and sensitivity toward Listeria monocytogenes with a limit of detection of 8.4 CFU/ml. CONCLUSIONS This work will focus on the development of the described phage-based Listeria monocytogenes biosensor.





Medical Technology and Devices

Cell Therapy & Tissue Engineering

Exploring the potential of bioengineered cell-based therapy for Parkinson's disease


Priyal Bagwe (1,2); D. Joshi (1,2); N. Chitre (2); A. Bansal (1,2); K. Murnane (2,3,4); and MJ D'Souza (1,2) 1. Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University 2. Department of Pharmaceutical Sciences, Mercer University College of Pharmacy 3. Department of Pharmacology, Toxicology & Neuroscience, School of Graduate Studies, Louisiana State University Health Sciences Center 4. Department of Psychiatry, School of Medicine, Louisiana State University Health Sciences Center BACKGROUND Parkinson's disease (PD) is the second most common neurodegenerative disorder, associated with decreased dopamine levels in the brain. The current treatments for Parkinson's disease include symptomatic relief with drugs. The goal of this study was to assess the potential of a regenerative medicine-based cell therapy approach to increase dopamine levels. In this study, we used rat adrenal pheochromocytoma (PC12) cells that can produce, store, and secrete dopamine. These cells were microencapsulated in the selectively permeable polymer membrane to protect them from immune responses. METHODS PC12 cells were suspended in sodium alginate solution containing trehalose. The cell suspension was sprayed using the modified spraying device through 0.7 mm nozzle into calcium chloride solution. The microcapsules were then transferred to chitosan glutamate solution to coat the alginate membrane. The microcapsules obtained were then transferred to the culture media and stored in the incubator. Efficiency of the microencapsulation process was calculated as the percentage of viable cells after the encapsulation process using acridine orange dye. The parameters affecting the size of the microcapsules i.e., flow rate, pump speed, nozzle diameter, and concentration of sodium alginate were varied to optimize the size of microcapsules. FTIR-ATR spectra of sodium alginate and the microcapsules were recorded. The stability of the microcapsules was assessed. Immuno-isolation and selective permeability of the microcapsules were confirmed via micro-BCA protein assay and dopamine release assay. The release of nitric oxide by antigen-presenting cells was determined by Griess's assay. RESULTS Efficiency of the microencapsulation process was found to be 81.57 ± 4.3 %. The size of microcapsules reduced with increasing flow rate, increasing pump speed and by decreasing the nozzle diameter and concentration of sodium alginate. The air flow rate of 350 L/h, pump speed of 9 RPM, nozzle diameter of 0.7 mm, and 0.5 % w/v solution of sodium alginate produced microcapsules of an average size of 38.2 µm. FTIR-ATR spectra confirmed the crosslinking of sodium alginate with calcium chloride. PC12 cells were viable inside the microcapsules suspended in media for 30 days. Dopamine release assay showed that the microencapsulated cells could release a consistent amount of dopamine for 30 days. Micro BCA protein assay showed that BSA could not cross the microcapsule membrane. This confirmed the selective permeability of the microcapsule membrane. Griess's assay showed that the PC12 cells were significantly more immunogenic as compared to microcapsules encapsulating PC12 cells. CONCLUSIONS We could successfully formulate microcapsules encapsulating live PC12 cells. The microcapsules were found to be stable, and encapsulated cells were viable inside the microcapsules. The microcapsules were selectively permeable, thus could provide adequate protection to encapsulated cells, and were non-immunogenic. Taken together, PC12 microcapsules thus have the potential to serve as an alternative treatment approach for PD.





 

Molecular and Biological Research

Employing Novel 3D Printed Oral Dissolving Films for COVID-19: Buccal Immunization of Microparticulate Vaccine Encapsulating the Spike S1 RBD Protein


Smital R. Patil, Sharon Vijayanand, Devyani Joshi, and Martin J. D'Souza Center for Drug Delivery Research, Vaccine Nanotechnology Laboratory, College of Pharmacy, Mercer University
BACKGROUND The development of an efficacious prophylactic measure for COVID-19 which can be available worldwide is highly critical. Current vaccines face some major challenges such as poor global distribution and availability due to the need for cold-chain storage. Moreover, these vaccines utilize the conventional, invasive intramuscular route of administration. Spike S1 receptor-binding domain (RBD) is highly immunogenic since it binds to the ACE-2 receptor leading to the entry of the virus into the host cell. We propose to formulate a microparticulate vaccine encapsulating RBD protein since microparticles (MPs) are highly stable and help in avoiding cold chain storage, thus improving their availability in developing countries. Further, incorporating these MPs in fast dissolving oral dissolving films (ODFs) will be beneficial in developing a patient compliant vaccine strategy with providing a robust systemic and mucosal immune response. Also, the use of 3D printing technology allows aseptic conditions for formulation and automation of the process. METHODS Microparticles were formulated using poly (lactic-co-glycolic) acid (PLGA) as polymer and spike S1 RBD protein as an antigen. CELLINK INKREDIBLE plus® 3D bioprinter was used for the formulation of fast dissolving ODFs using biodegradable polymers incorporating spike S1 protein MP and adjuvant (Alum + MF59) MP. ODFs were characterized for morphology and thickness using SEM. ODFs were characterized for diameter, weight variation, uniformity of thickness, disintegration time, and surface pH.
For in vivo studies, 6-8 weeks old Swiss Webster mice were utilized. The animals are administered with 1 prime and 2 booster doses of vaccine at weeks 0, 2, and 4 respectively. The serum samples were collected bi-weekly and analyzed for induction of humoral immune response as seen by production of total IgG, IgG subtypes, and IgA antibodies evaluated by ELISA. The immune organs collected post euthanizing were analyzed for the presence of CD4+ and CD8a T-lymphocytes. RESULTS We successfully formulated the MPs encapsulating Spike S1 RBD protein and adjuvant MPs. The yield of the MPs was >95%. The formulated ODFs were colorless, smooth, homogenous with an average diameter of 4.34 ± 0.094 mm. The ODFs with a thickness of 128.2 ± 0.032 µm were found to be disintegrated in the artificial saliva in 182 seconds. The surface pH was found to be 7.16. The in vivo studies exhibited induction of significantly higher humoral response demonstrated by the serum titers of IgG, IgG subtypes, and IgA. Cellular immune response was demonstrated by the presence of significantly higher levels of CD4+ and CD8a T lymphocytes. CONCLUSIONS

ODFs incorporated with RBD MPs were able to induce a humoral, mucosal, and cellular immune response. This formulation will be highly patient compliant and can be a potential vaccine candidate for COVID-19.




Microneedle vaccine induces cellular immunity and resistance to Neisseria gonorrhoeae infection in a murine model


Priyal Bagwe (1), L. Bajaj (1), RP Gala (2), S. Zughaier (3), and MJ D'Souza (1) 1. Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University
2. Fraunhofer USA, Center for Molecular Biotechnology
3. Qatar University, College of Medicine BACKGROUND There is a global rise in the number of cases of gonorrhea infection worldwide each year. Neisseria gonorrhoeae is the bacteria that causes gonorrhea infection and has gradually developed antimicrobial resistance. Due to antibiotic overuse for Covid-19 Coronavirus in 2020, the World Health Organization (WHO) has reported a record high rise in drug-resistant gonorrhea infection. Thus, there is an urgent need for an alternative therapy for gonorrhea. However, there is no vaccine for gonorrhea. This study investigates the induction of cellular immune responses and resistance to Neisseria gonorrhoeae infection upon immunization in a murine infection model. METHODS

Female mice were immunized intradermally with dissolving microneedles containing an inactivated whole-cell Neisseria gonorrhoeae microparticulate vaccine. The mice received one prime and two booster doses at two weeks intervals (weeks 0, 2, and 4). Enzyme-linked immunosorbent assay (ELISA) was used to measure the total IgG levels in the mice sera and IgA in vaginal washes. At week 6, the mice were challenged intravaginally using an established murine female genital tract infection model with live Neisseria gonorrhoeae. At the end of week 10, the mice were sacrificed, and their secondary immune organs such as spleen and lymph nodes were isolated to determine T-lymphocyte responses. The induction of T-cell immune response was assessed by analyzing CD4+ and CD8+ T-cell surface markers in the spleen and lymph nodes using flow cytometry.

RESULTS Serum immunoglobulin G (IgG) and vaginal immunoglobulin A (IgA) antibodies were generated against the antigen in groups receiving vaccine only and vaccine with adjuvants when compared to the untreated control group. The control groups cleared the infection in 10-12 days, whereas those immunized with our novel inactivated whole-cell vaccine cleared the infection in 6-9 days. Thus, significant protection was seen in the immunized mice. The mice receiving adjuvanted vaccine produced significantly higher CD4+ and CD8+ levels when compared to the untreated control group in the spleen and lymph nodes. CONCLUSIONS

The results demonstrated that humoral and cellular immunity to gonococcal infection could be induced by immunization with intradermal microneedles containing an inactivated gonorrhea antigen. Along with antibody response, cellular immune responses were also induced by the vaccine as demonstrated by the expression of CD4 and CD8. Therefore, this is an effective vaccination strategy. We will further study the levels of cross-protection in mice following a challenge with a different strain of Neisseria gonorrhoeae.




Dissolving microneedle embedded with microparticles: a novel pain free vaccine for respiratory syncytial virus


Ipshita Menon (1), S. Patil (1), SM Kang (2), and MJ D'Souza (1)
1. Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy
2. Georgia State University, Center for Inflammation, Immunity, and Infection
BACKGROUND Past vaccine trials for respiratory syncytial virus (RSV) using inactivated virus failed due to the antigen used. Thus, there is no vaccine for RSV. The fusion (F) protein integrated into a virus-like particle (VLP) was used as the antigen for this study. Here, the antigen was encapsulated in a biodegradable polymer to get a better antigen presentation. Furthermore, the skin provides an abundant presence of Langerhans cells, that can take up antigens and present them to the T cells. In this study, we embedded the microparticles in a dissolving microneedle patch to achieve a pain-free method of vaccination. METHODS

The vaccine and adjuvant microparticles were made using poly (lactic-co-glycolic acid) (PLGA) using high-pressure homogenization. Hyaluronic acid and trehalose were used to make the dissolving microneedles. The antigen integrity was tested using SDS-PAGE after formulation and post six months of storage at 4°C. The antigen presentation of the F-VLP microparticles with and without adjuvant monophosphoryl lipid A (MPL) microparticles was studied in vitro using DC 2.4 by analyzing the expression of major histocompatibility complex (MHC) molecules and autophagosomes using flow cytometry and fluorescence microscopy. Subsequently, the in vivo immunogenicity was studied by immunizing 6 to 8-week-old Swiss Webster mice with microparticle-loaded microneedles. The mice serum was collected periodically for further analysis. Next, the mice were challenged with wild-type RSV A2 virus (1x106/ mouse), and post challenge, the lungs of the mice were harvested. The mice serum was analyzed for the IgG, IgG2a, IgG1, and the lung homogenates were analyzed for IgA and the IgGs using ELISA.

RESULTS The optimized PLGA microparticles were in the range of 500-700 nm and had an encapsulation efficiency of 73% ± 10.5. The SDS PAGE verified the integrity of the F-VLP both post formulation and storage at 4 °C for eight months. The microparticle-loaded microneedles dissolved entirely within five minutes in murine skin. The H&E staining confirmed the formation of micropores on the stratum corneum. The in vitro testing showed an upregulation of MHC I and II in the cells treated with F-VLP microparticles with and without the adjuvant MPL microparticles. Similarly, the expression of autophagosomes was significantly high in the adjuvanted microparticle group compared to the F-VLP suspension. The in vivo testing showed significantly elevated IgG, IgG2a, and IgG1 in the serum and the lung homogenates of the mice. We also observed high IgA levels in the lung homogenates of the mice immunized with the adjuvanted microneedle vaccine. CONCLUSIONS

The F-VLP microparticle-loaded microneedles was able to induce a robust immune response. Thus, in the absence of a licensed vaccine, the microparticle-loaded microneedles will be a powerful tool for immunization for an infectious disease like RSV.




Zika vaccine microparticles-loaded dissolving microneedles induce a significant humoral immune response


Akanksha Kale and Martin D'Souza
Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University
BACKGROUND Zika is an infectious disease caused by the Zika virus. It has been associated with microcephaly, congenital Zika syndrome, and other birth defects. It also causes Guillain Barr Syndrome - an autoimmune disorder of the peripheral nervous system that culminates into paralysis and ultimately death. However, there is no approved treatment or vaccine available for Zika thus far. The current study explores polymeric microparticles (MPs) loaded in dissolving microneedles (MN) as a painless and needle-free vaccination strategy. METHODS

FDA-approved poly(lactic-co-glycolic) acid (PLGA) was selected to formulate vaccine MPs encapsulating inactivated Zika virus strain PRVABC59 via double emulsion solvent evaporation method. Adjuvant MPs encapsulating Alhydrogel® and MPL-A® were formulated separately following the similar method. Vaccine MPs were characterized for size, zeta potential, encapsulation efficiency, in vitro release, and antigen integrity. Morphology of the MPs was observed using a benchtop scanning electron microscope (SEM). In vitro immunogenicity of MPs in murine dendritic cells was evaluated using Griess assay and the ability to induce autophagosomes. In vitro cytotoxicity of MPs was evaluated MTT assay. Next, vaccine MPs with or without adjuvants were embedded in dissolving MN. These MN were characterized for length and dissolution time. The vaccine MPs with or without adjuvants were administered to Swiss Webster mice either via intramuscular (IM) injection or transdermal microneedles as one prime and two booster doses. Serum samples were collected periodically and analyzed for total IgG, IgG2a, and IgG1 antibody titers. A group of mice receiving no treatment was used as a control group.

RESULTS The size and zeta potential of vaccine MPs were 573.4±10.18 nm and -22.6±0.503 mV. SEM showed that the MPs were spherical. The encapsulation efficiency was 55-70%. In vitro release study showed that 50% of the encapsulated antigen is released within 24 hours with a sustained release of remaining antigen over 7 days. Antigen integrity was confirmed by SDS-PAGE. Vaccine MPs were found to be immunogenic and non-cytotoxic in vitro in murine dendritic cells. The average length of MN in the array was 400 µm, which dissolved 10 minutes after application in murine skin. Mice immunized with vaccine MPs with or without adjuvants produced significantly higher total IgG, IgG2a, and IgG1 antibody titers when compared to the untreated control group. Induction of IgG2a and IgG1 indicated the Th-1 and Th-2 mediated immune response. Antibody titers of mice immunized via IM injection were comparable to that of the mice immunized via transdermal dissolving MN. CONCLUSIONS

Zika vaccine MPs with sustained-release properties were formulated and were found immunogenic and non-cytotoxic in vitro. They induced a significant humoral immune response with both Th-1 and Th-2 mediated immune responses after IM and transdermal administration. Thus, the study established the feasibility of a potential transdermal Zika vaccine.




Needle-less, Pain-free Microneedle Immunization for COVID-19 using the Spike Glycoprotein Encapsulated in Microparticles Induces a Strong Humoral Response


Smital R. Patil, Sharon Vijayanand, Devyani Joshi, Keegan Braz Gomes, Ipshita J. Menon, and Martin J. D'Souza Center for Drug Delivery Research, Vaccine Nanotechnology Laboratory, College of Pharmacy, Mercer University BACKGROUND

COVID-19 has affected around 235 million people and caused more than 4.8 deaths worldwide. Current highly efficacious vaccines for COVID-19 pose the major drawback of the requirement of cold chain storage and administration through invasive routes such as intramuscular. The SARS-CoV spike glycoprotein is a viral epitope that is capable of inducing an immune response in the body and thus a suitable antigen for formulating a vaccine for COVID-19. Additionally, microparticles (MPs) are suitable delivery vehicles for vaccine antigens as they are better taken up by antigen-presenting cells, inducing a more robust immune response against the antigen. For this study, the Spike glycoprotein was loaded into poly (lactic-co-glycolic acid)(PLGA) MPs, which were then incorporated into dissolving microneedles, which have shown to be a promising delivery system for large molecules such as proteins. Also, microparticles and microneedles offer the added advantage of avoiding cold chain storage thus making their availability in developing countries possible. Thus, our vaccine will provide a non-invasive alternative for current COVID-19 vaccines.

METHODS

Formulation of Spike glycoprotein MPs was done using double emulsion method and PLGA as polymer. Similarly, adjuvant MPs for Alum and MF59 were also formulated. The MPs were subsequently lyophilized, characterized, and assessed for cytotoxicity, innate and adaptive immune response in vitro. The vaccine MPs were also assessed for expression of autophagosomes in dendritic cells (DC 2.4). Vaccine MPs were loaded into a hyaluronic acid gel and centrifuged to produce vaccine-loaded fast-dissolving microneedles. The efficacy of the vaccine microneedle patches was assessed in vivo in a preclinical murine model to induce a humoral immune response.

RESULTS Our vaccine MPs were successfully formulated and characterized for their size, surface charge, polydispersity index, and antigen encapsulation efficiency. The vaccine particles were found to be non-cytotoxic and induced a significantly higher nitrite production in mammalian cells compared to non-treated cells and cells treated with antigen unloaded microparticles. DC 2.4 pulsed with the vaccine microparticles also produced a significantly higher expression of antigen-presenting molecules: major histocompatibility complex I (MHC I), CD80, MHC II, and CD40 on the surface of the dendritic cells. DC 2.4 cells pulsed with vaccine plus adjuvant groups produced a significantly higher number of autophagosomes. Our vaccine formulation was able to induce significantly high levels of IgG, IgG1, IgG2a in a murine mouse model. CONCLUSIONS

Our formulated vaccine exhibited high immunogenicity in vitro and in vivo indicating a robust immune response. Thus, our vaccine has the potential to be a promising vaccine candidate in the ongoing COVID-19 pandemic the world is facing currently.




A Microneedle approach for a Virus-like-particle (VLP)-based Microparticulate Flu Vaccine


Sharon Vijayanand (1), K. Braz Gomes (1), SM Kang (2), & MJ D'Souza (1)
1. Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy
2. Georgia State University, Institute for Biomedical Sciences
BACKGROUND Influenza affects both adults and children every year and protection against infection can be achieved only with the yearly vaccine administration. Developing a cross-protective influenza vaccine is of prime importance, as it can better protect against a multitude of influenza subtypes, thereby reducing the frequency of immunization. In this study, we developed and tested a microparticulate (MP) vaccine for influenza, which utilizes tandem repeats of the highly conserved extracellular domain of the matrix-2 (M2e) protein, resulting in M2e virus-like particles (VLPs). Moreover, our vaccination strategy includes administration of the MP vaccine via the skin as pain-free dissolving microneedle (MN) patches. METHODS The antigen-loaded microparticles (MPs) were formulated by using a double-emulsion method and lyophilized. The M2e VLP microparticles were characterized for their yield, size, charge, polydispersity index (PDI), and encapsulation efficiency. In vitro analysis of the M2e VLP MPs included testing their ability to induce innate immune responses through nitrite production and antigen presentation on antigen-presenting cells (APC's) using flow cytometry. In vivo, the final vaccine containing M2e VLP and adjuvants Alhydrogel® + monophosphoryl-lipid-A (MPL-A) ® MPs was administered to mice through the skin using fast-dissolving microneedle (MN) patches. The immunization involved one prime and one booster dose administered three weeks apart. The mice sera were collected at various time points and ELISA was done to determine M2e-specific IgG levels. At Week 6 the mice were challenged with a live strain of influenza virus (A/Philippines/H3N2) and later sacrificed, having their lymph nodes, spleen, and lungs harvested for further analysis of immune markers such as CD4, CD8, and memory markers such as CD45R, CD62L, and CD27. RESULTS The results show that the MP vaccine induced a strong innate immune response in vitro. Additionally, IgG levels of the vaccine groups were higher compared to the M2e VLP in suspension. Similarly, flow cytometry analysis showed a higher percentage of spleen and lymph node cells expressing CD4 and CD8 in the microparticle-vaccinated mice as compared to the antigen in suspension. The results of the study indicate that the MP vaccine can induce effective humoral (IgG) as well as cell-mediated (CD4 and CD8) immune responses to protect against influenza. The assessment of memory markers, CD45R, and CD27 in the splenocytes showed a significant increase for the vaccine plus adjuvant group compared to the antigen suspension group. CONCLUSIONS The study suggests that the M2e VLP MP vaccine which was administered via the skin can induce an effective immune response. Transdermal delivery of the vaccine via dissolving microneedles is an attractive option as it will greatly increase patient compliance, particularly for toddlers and children. Further studies will be done to test the cross-protectiveness of the vaccine candidate using various assays.




A Minimally Invasive Microneedle Vaccine Patch for COVID-19


Sharon Vijayanand, SR Patil, D. Joshi, and MJ D'Souza
Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy
BACKGROUND The SARS COV-2 virus caused the 2020 pandemic and continues to mutate and infect humans. Yearly immunization against SARS CoV-2 and its mutant strains may be required. Here, we propose a microparticulate (MP) inactivated vaccine which can be administered as pain-free dissolving microneedles. Microneedle (MN) patches are attractive for vaccine delivery because mass immunization can be achieved immediately if the vaccine can be self-administered. The vaccine formulation utilizes an MP matrix formulated using poly (lactic-co-glycolic acid) (PLGA), a biodegradable polymer that encapsulates the antigen and provides improved antigen uptake in antigen-presenting cells (APCs).
METHODS In brief, the microparticles (MPs) were formulated using a double emulsion method and lyophilized. The ability of the vaccine MPs to elicit an in vitro innate immune response was assessed using a nitric oxide assay. The safety of the vaccine MP was assessed by performing an in vitro cytotoxicity study. The in vitro antigen presentation of the MP vaccine by the antigen-presenting cells (APCs) was assessed using flow cytometry. For in vivo assessment, the mice were immunized with the MP vaccine administered as dissolving MN patches. The immunization included one prime and two booster doses at weeks 0, 2, and 4 respectively. ELISA was done to assess the IgG level in mice sera at various time points. The animals were sacrificed at week 10 and their organs were collected and processed for further analysis and expression of immune markers. RESULTS The MPs were less than 1 micron in size. The Griess's nitrite assay showed a significantly higher (p<0.05) release of nitric oxide (NO) by antigen-presenting cells (APCs), in response to being stimulated by the vaccine, with and without adjuvants, as compared to untreated cells. The results obtained c=from the cytotoxicity study indicated that the vaccine MP is safe in low doses. Flow cytometry analysis confirms, significantly high (p<0.05) expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 respectively on the surface of APCs, in response to being pulsed with the MP with and without the adjuvant, as compared to untreated cells. ELISA was done to test the IgG levels in sera of immunized mice. The total IgG levels of the vaccine + adjuvant groups were significantly higher (p<0.05) than the group that received no treatment. CONCLUSIONS Based on the results, we summarize that the microparticulate vaccine can produce an effective innate and adaptive immune response when administered as an MN patch via the skin. The MP vaccine approach combined with the MN vaccination strategy is an effective immunization method. Moreover, MN patches allow self-administration which will decrease the need for trained pharmacists for immunization and greatly increase compliance with children as they are painless.





 

Nanotechnology

Employing Novel 3D Printed Oral Dissolving Films for COVID-19: Buccal Immunization of Microparticulate Vaccine Encapsulating the Spike S1 RBD Protein


Smital R. Patil, Sharon Vijayanand, Devyani Joshi, and Martin J. D'Souza Center for Drug Delivery Research, Vaccine Nanotechnology Laboratory, College of Pharmacy, Mercer University
BACKGROUND The development of an efficacious prophylactic measure for COVID-19 which can be available worldwide is highly critical. Current vaccines face some major challenges such as poor global distribution and availability due to the need for cold-chain storage. Moreover, these vaccines utilize the conventional, invasive intramuscular route of administration. Spike S1 receptor-binding domain (RBD) is highly immunogenic since it binds to the ACE-2 receptor leading to the entry of the virus into the host cell. We propose to formulate a microparticulate vaccine encapsulating RBD protein since microparticles (MPs) are highly stable and help in avoiding cold chain storage, thus improving their availability in developing countries. Further, incorporating these MPs in fast dissolving oral dissolving films (ODFs) will be beneficial in developing a patient compliant vaccine strategy with providing a robust systemic and mucosal immune response. Also, the use of 3D printing technology allows aseptic conditions for formulation and automation of the process. METHODS Microparticles were formulated using poly (lactic-co-glycolic) acid (PLGA) as polymer and spike S1 RBD protein as an antigen. CELLINK INKREDIBLE plus® 3D bioprinter was used for the formulation of fast dissolving ODFs using biodegradable polymers incorporating spike S1 protein MP and adjuvant (Alum + MF59) MP. ODFs were characterized for morphology and thickness using SEM. ODFs were characterized for diameter, weight variation, uniformity of thickness, disintegration time, and surface pH.
For in vivo studies, 6-8 weeks old Swiss Webster mice were utilized. The animals are administered with 1 prime and 2 booster doses of vaccine at weeks 0, 2, and 4 respectively. The serum samples were collected bi-weekly and analyzed for induction of humoral immune response as seen by production of total IgG, IgG subtypes, and IgA antibodies evaluated by ELISA. The immune organs collected post euthanizing were analyzed for the presence of CD4+ and CD8a T-lymphocytes. RESULTS We successfully formulated the MPs encapsulating Spike S1 RBD protein and adjuvant MPs. The yield of the MPs was >95%. The formulated ODFs were colorless, smooth, homogenous with an average diameter of 4.34 ± 0.094 mm. The ODFs with a thickness of 128.2 ± 0.032 µm were found to be disintegrated in the artificial saliva in 182 seconds. The surface pH was found to be 7.16. The in vivo studies exhibited induction of significantly higher humoral response demonstrated by the serum titers of IgG, IgG subtypes, and IgA. Cellular immune response was demonstrated by the presence of significantly higher levels of CD4+ and CD8a T lymphocytes. CONCLUSIONS

ODFs incorporated with RBD MPs were able to induce a humoral, mucosal, and cellular immune response. This formulation will be highly patient compliant and can be a potential vaccine candidate for COVID-19.




Microneedle vaccine induces cellular immunity and resistance to Neisseria gonorrhoeae infection in a murine model


Priyal Bagwe (1), L. Bajaj (1), RP Gala (2), S. Zughaier (3), and MJ D'Souza (1) 1. Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University
2. Fraunhofer USA, Center for Molecular Biotechnology
3. Qatar University, College of Medicine BACKGROUND There is a global rise in the number of cases of gonorrhea infection worldwide each year. Neisseria gonorrhoeae is the bacteria that causes gonorrhea infection and has gradually developed antimicrobial resistance. Due to antibiotic overuse for Covid-19 Coronavirus in 2020, the World Health Organization (WHO) has reported a record high rise in drug-resistant gonorrhea infection. Thus, there is an urgent need for an alternative therapy for gonorrhea. However, there is no vaccine for gonorrhea. This study investigates the induction of cellular immune responses and resistance to Neisseria gonorrhoeae infection upon immunization in a murine infection model. METHODS

Female mice were immunized intradermally with dissolving microneedles containing an inactivated whole-cell Neisseria gonorrhoeae microparticulate vaccine. The mice received one prime and two booster doses at two weeks intervals (weeks 0, 2, and 4). Enzyme-linked immunosorbent assay (ELISA) was used to measure the total IgG levels in the mice sera and IgA in vaginal washes. At week 6, the mice were challenged intravaginally using an established murine female genital tract infection model with live Neisseria gonorrhoeae. At the end of week 10, the mice were sacrificed, and their secondary immune organs such as spleen and lymph nodes were isolated to determine T-lymphocyte responses. The induction of T-cell immune response was assessed by analyzing CD4+ and CD8+ T-cell surface markers in the spleen and lymph nodes using flow cytometry.

RESULTS Serum immunoglobulin G (IgG) and vaginal immunoglobulin A (IgA) antibodies were generated against the antigen in groups receiving vaccine only and vaccine with adjuvants when compared to the untreated control group. The control groups cleared the infection in 10-12 days, whereas those immunized with our novel inactivated whole-cell vaccine cleared the infection in 6-9 days. Thus, significant protection was seen in the immunized mice. The mice receiving adjuvanted vaccine produced significantly higher CD4+ and CD8+ levels when compared to the untreated control group in the spleen and lymph nodes. CONCLUSIONS

The results demonstrated that humoral and cellular immunity to gonococcal infection could be induced by immunization with intradermal microneedles containing an inactivated gonorrhea antigen. Along with antibody response, cellular immune responses were also induced by the vaccine as demonstrated by the expression of CD4 and CD8. Therefore, this is an effective vaccination strategy. We will further study the levels of cross-protection in mice following a challenge with a different strain of Neisseria gonorrhoeae.




Dissolving microneedle embedded with microparticles: a novel pain free vaccine for respiratory syncytial virus


Ipshita Menon (1), S. Patil (1), SM Kang (2), and MJ D'Souza (1)
1. Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy
2. Georgia State University, Center for Inflammation, Immunity, and Infection
BACKGROUND Past vaccine trials for respiratory syncytial virus (RSV) using inactivated virus failed due to the antigen used. Thus, there is no vaccine for RSV. The fusion (F) protein integrated into a virus-like particle (VLP) was used as the antigen for this study. Here, the antigen was encapsulated in a biodegradable polymer to get a better antigen presentation. Furthermore, the skin provides an abundant presence of Langerhans cells, that can take up antigens and present them to the T cells. In this study, we embedded the microparticles in a dissolving microneedle patch to achieve a pain-free method of vaccination. METHODS

The vaccine and adjuvant microparticles were made using poly (lactic-co-glycolic acid) (PLGA) using high-pressure homogenization. Hyaluronic acid and trehalose were used to make the dissolving microneedles. The antigen integrity was tested using SDS-PAGE after formulation and post six months of storage at 4°C. The antigen presentation of the F-VLP microparticles with and without adjuvant monophosphoryl lipid A (MPL) microparticles was studied in vitro using DC 2.4 by analyzing the expression of major histocompatibility complex (MHC) molecules and autophagosomes using flow cytometry and fluorescence microscopy. Subsequently, the in vivo immunogenicity was studied by immunizing 6 to 8-week-old Swiss Webster mice with microparticle-loaded microneedles. The mice serum was collected periodically for further analysis. Next, the mice were challenged with wild-type RSV A2 virus (1x106/ mouse), and post challenge, the lungs of the mice were harvested. The mice serum was analyzed for the IgG, IgG2a, IgG1, and the lung homogenates were analyzed for IgA and the IgGs using ELISA.

RESULTS The optimized PLGA microparticles were in the range of 500-700 nm and had an encapsulation efficiency of 73% ± 10.5. The SDS PAGE verified the integrity of the F-VLP both post formulation and storage at 4 °C for eight months. The microparticle-loaded microneedles dissolved entirely within five minutes in murine skin. The H&E staining confirmed the formation of micropores on the stratum corneum. The in vitro testing showed an upregulation of MHC I and II in the cells treated with F-VLP microparticles with and without the adjuvant MPL microparticles. Similarly, the expression of autophagosomes was significantly high in the adjuvanted microparticle group compared to the F-VLP suspension. The in vivo testing showed significantly elevated IgG, IgG2a, and IgG1 in the serum and the lung homogenates of the mice. We also observed high IgA levels in the lung homogenates of the mice immunized with the adjuvanted microneedle vaccine. CONCLUSIONS

The F-VLP microparticle-loaded microneedles was able to induce a robust immune response. Thus, in the absence of a licensed vaccine, the microparticle-loaded microneedles will be a powerful tool for immunization for an infectious disease like RSV.




Zika vaccine microparticles-loaded dissolving microneedles induce a significant humoral immune response


Akanksha Kale and Martin D'Souza
Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy, Mercer University
BACKGROUND Zika is an infectious disease caused by the Zika virus. It has been associated with microcephaly, congenital Zika syndrome, and other birth defects. It also causes Guillain Barr Syndrome - an autoimmune disorder of the peripheral nervous system that culminates into paralysis and ultimately death. However, there is no approved treatment or vaccine available for Zika thus far. The current study explores polymeric microparticles (MPs) loaded in dissolving microneedles (MN) as a painless and needle-free vaccination strategy. METHODS

FDA-approved poly(lactic-co-glycolic) acid (PLGA) was selected to formulate vaccine MPs encapsulating inactivated Zika virus strain PRVABC59 via double emulsion solvent evaporation method. Adjuvant MPs encapsulating Alhydrogel® and MPL-A® were formulated separately following the similar method. Vaccine MPs were characterized for size, zeta potential, encapsulation efficiency, in vitro release, and antigen integrity. Morphology of the MPs was observed using a benchtop scanning electron microscope (SEM). In vitro immunogenicity of MPs in murine dendritic cells was evaluated using Griess assay and the ability to induce autophagosomes. In vitro cytotoxicity of MPs was evaluated MTT assay. Next, vaccine MPs with or without adjuvants were embedded in dissolving MN. These MN were characterized for length and dissolution time. The vaccine MPs with or without adjuvants were administered to Swiss Webster mice either via intramuscular (IM) injection or transdermal microneedles as one prime and two booster doses. Serum samples were collected periodically and analyzed for total IgG, IgG2a, and IgG1 antibody titers. A group of mice receiving no treatment was used as a control group.

RESULTS The size and zeta potential of vaccine MPs were 573.4±10.18 nm and -22.6±0.503 mV. SEM showed that the MPs were spherical. The encapsulation efficiency was 55-70%. In vitro release study showed that 50% of the encapsulated antigen is released within 24 hours with a sustained release of remaining antigen over 7 days. Antigen integrity was confirmed by SDS-PAGE. Vaccine MPs were found to be immunogenic and non-cytotoxic in vitro in murine dendritic cells. The average length of MN in the array was 400 µm, which dissolved 10 minutes after application in murine skin. Mice immunized with vaccine MPs with or without adjuvants produced significantly higher total IgG, IgG2a, and IgG1 antibody titers when compared to the untreated control group. Induction of IgG2a and IgG1 indicated the Th-1 and Th-2 mediated immune response. Antibody titers of mice immunized via IM injection were comparable to that of the mice immunized via transdermal dissolving MN. CONCLUSIONS

Zika vaccine MPs with sustained-release properties were formulated and were found immunogenic and non-cytotoxic in vitro. They induced a significant humoral immune response with both Th-1 and Th-2 mediated immune responses after IM and transdermal administration. Thus, the study established the feasibility of a potential transdermal Zika vaccine.




Needle-less, Pain-free Microneedle Immunization for COVID-19 using the Spike Glycoprotein Encapsulated in Microparticles Induces a Strong Humoral Response


Smital R. Patil, Sharon Vijayanand, Devyani Joshi, Keegan Braz Gomes, Ipshita J. Menon, and Martin J. D'Souza Center for Drug Delivery Research, Vaccine Nanotechnology Laboratory, College of Pharmacy, Mercer University BACKGROUND

COVID-19 has affected around 235 million people and caused more than 4.8 deaths worldwide. Current highly efficacious vaccines for COVID-19 pose the major drawback of the requirement of cold chain storage and administration through invasive routes such as intramuscular. The SARS-CoV spike glycoprotein is a viral epitope that is capable of inducing an immune response in the body and thus a suitable antigen for formulating a vaccine for COVID-19. Additionally, microparticles (MPs) are suitable delivery vehicles for vaccine antigens as they are better taken up by antigen-presenting cells, inducing a more robust immune response against the antigen. For this study, the Spike glycoprotein was loaded into poly (lactic-co-glycolic acid)(PLGA) MPs, which were then incorporated into dissolving microneedles, which have shown to be a promising delivery system for large molecules such as proteins. Also, microparticles and microneedles offer the added advantage of avoiding cold chain storage thus making their availability in developing countries possible. Thus, our vaccine will provide a non-invasive alternative for current COVID-19 vaccines.

METHODS

Formulation of Spike glycoprotein MPs was done using double emulsion method and PLGA as polymer. Similarly, adjuvant MPs for Alum and MF59 were also formulated. The MPs were subsequently lyophilized, characterized, and assessed for cytotoxicity, innate and adaptive immune response in vitro. The vaccine MPs were also assessed for expression of autophagosomes in dendritic cells (DC 2.4). Vaccine MPs were loaded into a hyaluronic acid gel and centrifuged to produce vaccine-loaded fast-dissolving microneedles. The efficacy of the vaccine microneedle patches was assessed in vivo in a preclinical murine model to induce a humoral immune response.

RESULTS Our vaccine MPs were successfully formulated and characterized for their size, surface charge, polydispersity index, and antigen encapsulation efficiency. The vaccine particles were found to be non-cytotoxic and induced a significantly higher nitrite production in mammalian cells compared to non-treated cells and cells treated with antigen unloaded microparticles. DC 2.4 pulsed with the vaccine microparticles also produced a significantly higher expression of antigen-presenting molecules: major histocompatibility complex I (MHC I), CD80, MHC II, and CD40 on the surface of the dendritic cells. DC 2.4 cells pulsed with vaccine plus adjuvant groups produced a significantly higher number of autophagosomes. Our vaccine formulation was able to induce significantly high levels of IgG, IgG1, IgG2a in a murine mouse model. CONCLUSIONS

Our formulated vaccine exhibited high immunogenicity in vitro and in vivo indicating a robust immune response. Thus, our vaccine has the potential to be a promising vaccine candidate in the ongoing COVID-19 pandemic the world is facing currently.




A Microneedle approach for a Virus-like-particle (VLP)-based Microparticulate Flu Vaccine


Sharon Vijayanand (1), K. Braz Gomes (1), SM Kang (2), & MJ D'Souza (1)
1. Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy
2. Georgia State University, Institute for Biomedical Sciences
BACKGROUND Influenza affects both adults and children every year and protection against infection can be achieved only with the yearly vaccine administration. Developing a cross-protective influenza vaccine is of prime importance, as it can better protect against a multitude of influenza subtypes, thereby reducing the frequency of immunization. In this study, we developed and tested a microparticulate (MP) vaccine for influenza, which utilizes tandem repeats of the highly conserved extracellular domain of the matrix-2 (M2e) protein, resulting in M2e virus-like particles (VLPs). Moreover, our vaccination strategy includes administration of the MP vaccine via the skin as pain-free dissolving microneedle (MN) patches. METHODS The antigen-loaded microparticles (MPs) were formulated by using a double-emulsion method and lyophilized. The M2e VLP microparticles were characterized for their yield, size, charge, polydispersity index (PDI), and encapsulation efficiency. In vitro analysis of the M2e VLP MPs included testing their ability to induce innate immune responses through nitrite production and antigen presentation on antigen-presenting cells (APC's) using flow cytometry. In vivo, the final vaccine containing M2e VLP and adjuvants Alhydrogel® + monophosphoryl-lipid-A (MPL-A) ® MPs was administered to mice through the skin using fast-dissolving microneedle (MN) patches. The immunization involved one prime and one booster dose administered three weeks apart. The mice sera were collected at various time points and ELISA was done to determine M2e-specific IgG levels. At Week 6 the mice were challenged with a live strain of influenza virus (A/Philippines/H3N2) and later sacrificed, having their lymph nodes, spleen, and lungs harvested for further analysis of immune markers such as CD4, CD8, and memory markers such as CD45R, CD62L, and CD27. RESULTS The results show that the MP vaccine induced a strong innate immune response in vitro. Additionally, IgG levels of the vaccine groups were higher compared to the M2e VLP in suspension. Similarly, flow cytometry analysis showed a higher percentage of spleen and lymph node cells expressing CD4 and CD8 in the microparticle-vaccinated mice as compared to the antigen in suspension. The results of the study indicate that the MP vaccine can induce effective humoral (IgG) as well as cell-mediated (CD4 and CD8) immune responses to protect against influenza. The assessment of memory markers, CD45R, and CD27 in the splenocytes showed a significant increase for the vaccine plus adjuvant group compared to the antigen suspension group. CONCLUSIONS The study suggests that the M2e VLP MP vaccine which was administered via the skin can induce an effective immune response. Transdermal delivery of the vaccine via dissolving microneedles is an attractive option as it will greatly increase patient compliance, particularly for toddlers and children. Further studies will be done to test the cross-protectiveness of the vaccine candidate using various assays.




A Minimally Invasive Microneedle Vaccine Patch for COVID-19


Sharon Vijayanand, SR Patil, D. Joshi, and MJ D'Souza
Mercer University, Vaccine Nanotechnology Laboratory, Center for Drug Delivery Research, College of Pharmacy
BACKGROUND The SARS COV-2 virus caused the 2020 pandemic and continues to mutate and infect humans. Yearly immunization against SARS CoV-2 and its mutant strains may be required. Here, we propose a microparticulate (MP) inactivated vaccine which can be administered as pain-free dissolving microneedles. Microneedle (MN) patches are attractive for vaccine delivery because mass immunization can be achieved immediately if the vaccine can be self-administered. The vaccine formulation utilizes an MP matrix formulated using poly (lactic-co-glycolic acid) (PLGA), a biodegradable polymer that encapsulates the antigen and provides improved antigen uptake in antigen-presenting cells (APCs).
METHODS In brief, the microparticles (MPs) were formulated using a double emulsion method and lyophilized. The ability of the vaccine MPs to elicit an in vitro innate immune response was assessed using a nitric oxide assay. The safety of the vaccine MP was assessed by performing an in vitro cytotoxicity study. The in vitro antigen presentation of the MP vaccine by the antigen-presenting cells (APCs) was assessed using flow cytometry. For in vivo assessment, the mice were immunized with the MP vaccine administered as dissolving MN patches. The immunization included one prime and two booster doses at weeks 0, 2, and 4 respectively. ELISA was done to assess the IgG level in mice sera at various time points. The animals were sacrificed at week 10 and their organs were collected and processed for further analysis and expression of immune markers. RESULTS The MPs were less than 1 micron in size. The Griess's nitrite assay showed a significantly higher (p<0.05) release of nitric oxide (NO) by antigen-presenting cells (APCs), in response to being stimulated by the vaccine, with and without adjuvants, as compared to untreated cells. The results obtained c=from the cytotoxicity study indicated that the vaccine MP is safe in low doses. Flow cytometry analysis confirms, significantly high (p<0.05) expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 respectively on the surface of APCs, in response to being pulsed with the MP with and without the adjuvant, as compared to untreated cells. ELISA was done to test the IgG levels in sera of immunized mice. The total IgG levels of the vaccine + adjuvant groups were significantly higher (p<0.05) than the group that received no treatment. CONCLUSIONS Based on the results, we summarize that the microparticulate vaccine can produce an effective innate and adaptive immune response when administered as an MN patch via the skin. The MP vaccine approach combined with the MN vaccination strategy is an effective immunization method. Moreover, MN patches allow self-administration which will decrease the need for trained pharmacists for immunization and greatly increase compliance with children as they are painless.