Electroporation of tissue and cells for drug delivery applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1994.Includes bibliographical references (leaves 245-262).by Mark R. Prausnitz.Ph.D

Coated microneedles for transdermal delivery,

Abstract Coated microneedles have been shown to deliver proteins and DNA into the skin in a minimally invasive manner. However, detailed studies examining coating methods and their breadth of applicability are lacking. This study's goal was to develop a simple, versatile and controlled microneedle coating process to make uniform coatings on microneedles and establish the breadth of molecules and particles that can be coated onto microneedles. First, microneedles were fabricated from stainless steel sheets as single microneedles or arrays of microneedles. Next, a novel micron-scale dip-coating process and a GRAS coating formulation were designed to reliably produce uniform coatings on both individual and arrays of microneedles. This process was used to coat compounds including calcein, vitamin B, bovine serum albumin and plasmid DNA. Modified vaccinia virus and microparticles of 1 to 20 μm diameter were also coated. Coatings could be localized just to the needle shafts and formulated to dissolve within 20 s in porcine cadaver skin. Histological examination validated that microneedle coatings were delivered into the skin and did not wipe off during insertion. In conclusion, this study presents a simple, versatile, and controllable method to coat microneedles with proteins, DNA, viruses and microparticles for rapid delivery into the skin

Effect of Osmotic Pressure on the Stability of Whole Inactivated Influenza Vaccine for Coating on Microneedles

Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 – 6×10−4 cm s–1) and high Arrhenius activation energy (Ea = 15.0 kcal mol–1), indicating that the water molecules diffused through the viral lipid membranes. After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination

Self‐healing encapsulation and controlled release of vaccine antigens from PLGA microparticles delivered by microneedle patches

There is an urgent need to reduce reliance on hypodermic injections for many vaccines to increase vaccination safety and coverage. Alternative approaches include controlled release formulations, which reduce dosing frequencies, and utilizing alternative delivery devices such as microneedle patches (MNPs). This work explores development of controlled release microparticles made of poly (lactic‐co‐glycolic acid) (PLGA) that stably encapsulate various antigens though aqueous active self‐healing encapsulation (ASE). These microparticles are incorporated into rapid‐dissolving MNPs for intradermal vaccination.PLGA microparticles containing Alhydrogel are loaded with antigens separate from microparticle fabrication using ASE. This avoids antigen expsoure to many stressors. The microparticles demonstrate bi‐phasic release, with initial burst of soluble antigen, followed by delayed release of Alhydrogel‐complexed antigen over approximately 2 months in vitro. For delivery, the microparticles are incorporated into MNPs designed with pedestals to extend functional microneedle length. These microneedles readily penetrate skin and rapidly dissolve to deposit microparticles intradermally. Microparticles remain in the tissue for extended residence, with MNP‐induced micropores resealing readily. In animal models, these patches generate robust immune responses that are comparable to conventional administration techniques. This lays the framework for a versatile vaccine delivery system that could be self‐applied with important logistical advantages over hypodermic injections.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147859/1/btm210103-sup-0001-supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147859/2/btm210103_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147859/3/btm210103.pd

Water permeation through stratum corneum lipid bilayers from atomistic simulations

Stratum corneum, the outermost layer of skin, consists of keratin filled rigid non-viable corneocyte cells surrounded by multilayers of lipids. The lipid layer is responsible for the barrier properties of the skin. We calculate the excess chemical potential and diffusivity of water as a function of depth in lipid bilayers with compositions representative of the stratum corneum using atomistic molecular dynamics simulations. The maximum in the excess free energy of water inside the lipid bilayers is found to be twice that of water in phospholipid bilayers at the same temperature. Permeability, which decreases exponentially with the free energy barrier, is reduced by several orders of magnitude as compared to with phospholipid bilayers. The average time it takes for a water molecule to cross the bilayer is calculated by solving the Smoluchowski equation in presence of the free energy barrier. For a bilayer composed of a 2:2:1 molar ratio of ceramide NS 24:0, cholesterol and free fatty acid 24:0 at 300K, we estimate the permeability P=3.7e-9 cm/s and the average crossing time \tau_{av}=0.69 ms. The permeability is about 30 times smaller than existing experimental results on mammalian skin sections.Comment: latex, 8 pages, 6 figure

Enhanced Immune Responses Conferring Cross-Protection by Skin Vaccination With a Tri-Component Influenza Vaccine Using a Microneedle Patch

Skin vaccination using biodegradable microneedle patch (MNP) technology in vaccine delivery is a promising strategy showing significant advantages over conventional flu shots. In this study, we developed an MNP encapsulating a 4M2e-tFliC fusion protein and two types of whole inactivated influenza virus vaccines (H1N1 and H3N2) as a universal vaccine candidate. We demonstrated that mice receiving this tri-component influenza vaccine via MNP acquired improved IgG1 antibody responses with more balanced IgG1/IgG2a antibody responses and enhanced cellular immune responses, including increased populations of IL-4 and IFN-γ producing cells and higher frequencies of antigen-specific plasma cells compared with intramuscular injection. In addition, stronger germinal center reactions, increased numbers of Langerin-positive migratory dendritic cells, and increased cytokine secretion were observed in the skin-draining lymph nodes after immunization with the tri-component influenza MNP vaccine. The MNP-immunized group also possessed enhanced protection against a heterologous reassortant A/Shanghai/2013 H7N9 (rSH) influenza virus infection. Furthermore, the sera collected from 4M2e-tFliC MNP-immunized mice were demonstrated to have antiviral efficacy against reassortant A/Vietnam/1203/2004 H5N1 (rVet) and A/Shanghai/2013 H7N9 (rSH) virus challenges. The immunological advantages of skin vaccination with this tri-component MNP vaccine could offer a promising approach to develop an easily applicable and broadly protective universal influenza vaccine

Enhanced Immune Responses by Skin Vaccination with Influenza Subunit Vaccine in Young Hosts

Skin has gained substantial attention as a vaccine target organ due to its immunological properties, which include a high density of professional antigen presenting cells (APCs). Previous studies have demonstrated the effectiveness of this vaccination route not only in animal models but also in adults. Young children represent a population group that is at high risk from influenza infection. As a result, this group could ben- efit significantly from influenza vaccine delivery approaches through the skin and the improved immune response it can induce. In this study, we compared the immune responses in young BALB/c mice upon skin delivery of influenza vaccine with vaccination by the conventional intramuscular route. Young mice that received 5 fLg of H1N1 A/Ca/07/09 influenza subunit vaccine using MN demonstrated an improved serum antibody response (IgG1 and IgG2a) when compared to the young IM group, accompanied by higher numbers of influenza-specific antibody secreting cells (ASCs) in the bone marrow. In addition, we observed increased activation of follicular helper T cells and formation of germinal centers in the regional lymph nodes in the MN immunized group, rapid clearance of the virus from their lungs as well as complete survival, compared with partial protection observed in the IM-vaccinated group. Our results support the hypothesis that influenza vaccine delivery through the skin would be beneficial for protecting the high-risk young population from influenza infection

Rapid local anesthesia in humans using minimally invasive microneedles

Objective: This study tested the hypothesis that minimally invasive microneedles cause less pain during injection of lidocaine, but induce local anesthesia in humans with the same rapid onset and efficacy as intradermal lidocaine injection using hypodermic needles. Methods: This study was a randomized, single-blinded, within participants, controlled design. Hollow, 500-mm long microneedles were used to inject lidocaine to the forearm of 15 human participants. The associated pain was recorded using a visual analog (VAS) scale. The area and depth of numbness were determined at 0, 7.5, and 15 minutes after injection. Lidocaine was also injected to the dorsum of the hand near a vein, followed by placement of an intravenous catheter and measurement of associated pain. A 26-gauge intradermal bevel hypodermic needle similarly administered lidocaine on the opposite forearm/hand to serve as the positive control. Results: VAS pain scores revealed that injection using microneedles was significantly less painful than hypodermic needles for both the forearm and dorsum of the hand injections. However, there was no significant difference in the area or depth of the resulting numbness between the 2 treatment methods at any time point (0, 7.5, and 15 min) indicating that microneedles had immediate onset and were as effective as hypodermic needles in inducing dermal anesthesia. Moreover, insertion of an intravenous catheter immediately after lidocaine injection on the dorsum of the hand led to comparable pain scores for the microneedle and hypodermic needle treated sites, further confirming efficacy of microneedles in inducing rapid local anesthesia. Lastly, 77% of the participants preferred microneedles and 80% indicated that they did not consider microneedles to be painful. Discussion: This study demonstrates for the first time that microneedle-based lidocaine injection is as rapid and as effective as hypodermic injection in inducing local anesthesia while resulting in significantly less pain during injection