Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable

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Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles Abhay U. Andar, Ram Karan, Wolf T. Pecher, Priya DasSarma, William D. Hedrich, Audra L. Stinchcomb, and Shiladitya DasSarma Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00859 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Molecular Pharmaceutics

Microneedle-Assisted Skin Permeation by Non-toxic Bioengineerable Gas Vesicle Nanoparticles Abhay U. Andar†∞, Ram Karan‡∞, Wolf T. Pecher‡§∞, Priya DasSarma‡, William D. Hedrich†, Audra L. Stinchcomb*† and Shiladitya DasSarma*‡ †

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland,

Baltimore, MD 21201. ‡

Department of Microbiology and Immunology, School of Medicine, and Institute of Marine and

Environmental Technology, University System of Maryland, Baltimore, MD 21202 §Yale

Gordon College of Arts and Sciences, University of Baltimore, Baltimore, MD 21201

∞Authors

contributed equally to this work.

∗Professor

Audra Stinchcomb: Tel: 410-706-2646; email: [email protected] and

Professor Shiladitya DasSarma: Tel: 410-234-8847; email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm. In vitro diffusion studies using Yucatan miniature pig skin showed GVNP permeation to be enhanced after MN-treatment compared to untreated skin. GVNPs were found to be non-toxic to mammalian cells (human kidney and rat mycocardial myoblasts). These findings support the use of GVNPs as DDS for intradermal/transdermal permeation of protein- and peptide-based drugs. KEYWORDS Gas vesicle nanoparticles, Halobacterium, microneedles, in-vitro, Yucatan minipig skin


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INTRODUCTION Proteins and peptides have been considered plausible therapeutic modalities to combat human disease since the mid 1900’s and have gained importance as therapeutic agents in the past decade1. Thus far over 200 different types of protein and peptide-based drugs have been approved by the US Food and Drug Administration (FDA) for a variety of therapeutic applications for human diseases1–3. However, many proteins and peptides show insufficient efficacy when used in vivo and give unsatisfactory pharmacokinetic profiles due to inadequate shelf-life (stability), poor penetration across biological barrier membranes, short half-life in plasma, limited bioavailability, or immunogenic properties1,4,5. It is predicted that for drug delivery, nano-vehicles or carriers could be used to protect the therapeutic protein/peptide, maintain stability and activity, and improve biodistribution, while avoiding undesired side effects6–8. In an ideal setting, nano-vehicles should also decrease the drug clearance rate from the host system, meaning that lower concentrations of drugs need to be administered, thereby reducing the dosage, administration frequency and potential toxicity6–8. In this study, we utilized novel protein nanoparticles from an extremely salt-loving (halophilic) microbe for possible use as a DDS for delivering therapeutic peptides9. Gas vesicle nanoparticles (GVNPs) from Halobacterium sp. NRC-110, a well-characterized halophilic Archaeon growing in nearly saturated brine, are unlike other currently available or existing DDS (e.g. lipid vesicles, polymeric vesicles, colloidal nanoparticles, or ghost cells)6–8. GVNPs are easily purified by flotation, have a spindle-shaped structure made up purely of an extremely stable and rigid protein membrane, and range from 75-500 nm in length11–16. GVNPs have been found to be biocompatible for antigen delivery in mice and are processed slowly by antigen presenting cells17–19. Moreover, GVNPs may be bioengineered to display a variety of peptides and proteins


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with the potential for use in pharmaceutical applications9,20–23. Halobacterium sp. NRC-1 in particular, is being utilized to produce bioengineered GVNPs that possess qualities favorable to facilitate drug delivery9. A gene cluster with 14 gvp genes has been identified, most of which appear to be essential for GVNP production15. The GVNP membrane is made up of mainly of two proteins, GvpA and GvpC, and about five additional minor proteins13,14. GvpA is the major structural protein, with GvpC being bound to the external surface of the nanoparticles and stabilizing the structure24. A genetic system for protein display by fusion of the target protein to GvpC has been developed12,19. One of the major challenges in DDS has been the penetration of nano-carriers across the epidermis, especially for hydrophilic particles larger than 100 nm size. McAllister et al.25 using a “poke and patch” method on human cadaver epidermis found skin permeability to be inversely proportional to the molecular size of macromolecules, and passive diffusion through skin limited to 50-100 nm size nanoparticles. Other studies have shown that 200 nm liposomes are able to permeate through porcine skin26,27. Among larger nanoparticles, poly(lactic-co-glycolic acid) (PLGA) particles of 122-860 nm diameter have been shown to permeate skin through the follicular mechanism28. The surface chemistry and shape of nanoparticles are likely to be contributing factors to particle permeation through the skin29,30. The reported size of GVNPs suggested that these nanoparticles may be above the acceptable limit for passive transdermal delivery. RESULTS AND DISCUSSION In order to establish the precise size of GVNPs in preparations from Halobacterium sp. NRC-1, we used dynamic light scattering analysis employing a ZSPnano Zetasizer (Malvern Inc.,


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Malvern, UK). This confirmed that the majority of nanoparticles are in the range of ~200-400 nm average diameter, consistent with reported values11,13,14. The mean diameter of GVNPs in this study was found to be about 245 nm and the polydispersity index (PDI) was 0.12 (Figure 1), and therefore nearly monodispersed. Since dermal delivery of molecules larger than 100 nm was generally challenging, we expected that skin permeation of GVNPs would require or be enhanced by either physical or chemical enhancement techniques25,31. To facilitate the transport of GVNPs across the skin, we used a microneedle-assisted approach (Figure 2). The micropores created by microneedle (MN) treatment are known to facilitate transport of molecules via micron-scale hydrophilic aqueous conduits in the stratum corneum, the top-most layer of the skin. To determine the effects of MN-treatment, we carried out in vitro studies using full thickness Yucatan miniature pig skin (1.4-1.8 mm thick). Skin was placed in a 0.95 cm2 horizontal In-Line flow-through diffusion cell system (PermeGear, Hellertown, PA, USA) to establish the efficacy of skin permeation by purified GVNPs. Skin was treated with stainless steel 5 MN in plane arrays (750 µm long, 200 µm wide, 75 µm thick and 1.35 mm interneedle spacing), treated to provide 50 micropores to facilitate GVNP permeation. The receiver (sample) solution contained phosphate buffered saline, pH 7.4 (PBS) and was collected at 4 h intervals for a total of 24 h (Figure 3). The permeation of GVNPs was determined by using an immunoassay for GvpA, the major GVNP membrane protein, in receiver solution. Known quantities of GVNPs serially diluted were used directly as standards. We observed an increase in the amount of GVNPs that permeated through MN-treated skin over time, equivalent to 0.22 µg of GVNPs after 24 h (based on the GvpA immunoassay, Figure 3). A 3.3-fold increase in the rate of penetration of GVNPs was found compared to the MN-untreated skin. It is noteworthy that the GvpA protein cannot be


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measured in a free or soluble form without the use of a combination of extremely strong denaturing agents, and therefore, the immunoassays represent excellent evidence for the presence of the GVNPs themselves. In conjunction with the diffusion assay, we prepared skin samples for fluorescent confocal microscopy to visualize nuclei and permeated GVNPs using optimized confocal staining techniques29. DAPI was used for staining nuclei (blue) and GvpA antisera14 followed by fluorescence-tagged anti-rabbit antibodies (red) were used for detection of GVNPs. The GVNPs were clearly observed as brightly staining areas at the site of MN punctures and within the epidermis, after 24 hours (Figure 4). In negative controls without MN-treatment or in the absence of GVNP application, little or no GVNPs were detectable, confirming that the permeation of GVNPs across skin is enhanced by microneedles. The potential use of GVNPs as DDS is dependent on their biocompatibility and lack of toxicity. To address this concern, we utilized human kidney (HEK293) and rat myoblasts (H9c2(2-1)) cells to test GVNP toxicity employing an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. Purified GVNPs were used at 4 different concentrations (from 62.5 - 500 µg/mL of GVNPs, suspended in PBS, pH 7.4) and incubated for 24 h at 37°C. Cytotoxicity data revealed nearly 100% cell viability, up to the highest concentrations of GVNP tested (equivalent to 500 µg/mL GvpA), on both HEK293 and H9c2 cells (Figure 5). These results are consistent with past work where immunization of mice with GVNPs also had shown no adverse effects either at the injection site or on animal survival19,32–35. In this report, we have demonstrated for the first time, MN-assisted transdermal delivery of GVNPs. This finding coupled with our ability to bioengineer the nanoparticles to display


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antigenic proteins and enzymes shows the potential of GVNPs for use as a DDS and facilitating and improving the bioavailability of therapeutic protein/peptide conjugates. We confirmed the size of GVNPs to be ca. 245 nm and monodispersed using dynamic light scattering and documented the enhanced permeation of GVNPs through MN-treated skin based on the extent of GvpA protein measured by immunoblotting. The MN-directed permeation of GVNPs was also confirmed by visualization using fluorescent confocal microscopy. Gas vesicle nanoparticles (GVNPs) from Halobacterium sp. NRC-1 are unlike other currently available or existing nano-carriers6–9. The GVNP structure is made up purely of an extremely stable, rigid protein membrane and maintains its integrity for an extended period of time in dry form or as aqueous suspensions in the absence of refrigeration9,12–14,32,33. The ability to express arrays of foreign proteins as a part of the GVNP membrane via GvpC fusion, and their biocompatibility and lack of toxicity are other desirable qualities12,32,33,35. A number of studies have previously utilized GVNPs for therapeutics, antigen display and vaccine development 9,20–23 and the work described here extends the potential of these bioengineered nanoparticles for the delivery of displayed recombinant proteins using microneedles. Further investigations are required for understanding the fate of GVNPs in the epidermis and beyond. While there is the potential for breakdown of GVNPs after MN-assisted delivery, the stability of the structure and the slow processing previously reported17–19 suggest full retention of their integrity. Future studies using techniques such as transmission or scanning electron microscopy may be valuable for establishing the status of nanoparticles post-diffusion. The availability of GVNPs displaying luciferase also provides an assay for studies directed at the fate of the nanoparticles in vivo in the future17,34. The recent report of a protein in the interior of GVNPs 32 may allow the development of encapsulated therapeutic proteins, in addition to


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proteins displayed on the external surface of the structures. Such studies represent exciting opportunities for full realization of the potential of the approach, with MN-assisted delivery representing an enhancement. Halobacterium sp. NRC-1 exhibits a number of useful characteristics as microbial cell factories for recombinant protein production, including ease of culture, high production of recombinant proteins, and simple lysis, in addition to the presence of bioengineerable GVNPs that can be harvested by flotation36,37. These archaeal cells also lack toxic lipopolysaccharides commonly found in Gram-negative bacteria, making them desirable for production of specific protein/peptide therapeutic agents display on nanoparticles9. Therefore, production of recombinant proteins in Halobacterium offers an alternative to commonly used hosts, with potential advantages such as high level of expression, improved stability and solubility, and facility of purification34,36–39. In summary, we have demonstrated for the first time that microneedles may be used for transdermal permeation of Halobacterium GVNPs. This translational technology can be produced in a good manufacturing practice (GMP) facility. The stainless-steel MNs are manufactured in a clean-room and are usually autoclaved prior to their use in the clinic/animal studies40. As for the GVNPs, post cell-lysis, these robust and versatile nanoparticles may be filter purified using conventional filtration techniques. Future studies are needed to determine whether this approach will provide an alternative method for transdermal drug delivery of therapeutic proteins and peptides arrayed on GVNPs. EXPERIMENTAL SECTION GVNP harvest. The processes for producing and harvesting GVNPs have been previously


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described12. Briefly, Halobacterium sp. strain NRC-1 (ATCC 700922/JCM11081)10 lawns were lysed by addition of PBS solution [137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, and 2 mM potassium phosphate monobasic (pH 7.4)] containing 10 mM MgSO4 and 10 µg/mL of DNase I (Roche Diagnostics, Indianapolis, IN) and the cell lysate suspension was incubated for 3 h at 37°C. Lysates were then centrifuged at 60 x g overnight in a swinging bucket rotor in a Jouan CR412 centrifuge (Thermo Scientific, Rockford, IL) to accelerate floatation of the gas-filled nanoparticles. Next, intact buoyant nanoparticles were carefully collected into fresh tubes and re-suspended in PBS solution, floated by overnight centrifugation and harvested. The floatation procedure was repeated until a white, milky GVNP suspension was obtained. The total amount of GVNP was quantified as previously described14. Particle characterization. Dynamic light scattering of GVNP suspensions was analyzed in triplicate using a ZSPNano Zetasizer (Malvern Inc., Malvern, UK). The particle diameter and polydispersity of GVNP populations were determined, and the size distribution values were represented as mean diameter and polydispersity index (PDI). In vitro permeation experiments. Yucatan miniature pig skin (Sinclair Bio Resources, LLC) of full thickness, harvested from the dorsal region of a 9 month old animal, was used for this study. The subcutaneous fat was separated from the skin samples using a scalpel and any sparse hair on the skin was clipped. Skin samples were immediately stored at -20°C until further use. Before each permeation study, skin samples were allowed to thaw for approximately 30 min. Skin was dermatomed to a thickness of about 1.4-1.8 mm. The dermis thickness was evaluated using calipers and the skin placed on a 3-4 mm thick polydimethylsiloxane (PDMS) base in a petridish. PDMS is a silicone polymer which helps mimic the natural mechanical support of the


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underlying tissue/dermis because of its comparable structural elasticity. The Prausnitz laboratory MN array was fabricated by laser-cutting stainless steel sheets as reported previously25. The MN array was constructed of solid stainless steel, with two-dimensional ‘in-plane’ rows, with ten rows each consisting of five microneedles, oriented with their axes parallel to the steel sheets. The needles were 750 µm long, 200 µm wide, 75 µm thick and spaced 1.35 mm apart. MNs were inserted into the skin manually by applying gentle pressure to the skin, creating 50 micropores in the diffusion area (0.95 cm2). MN skin treatment was performed as previously described41. The MN-treated skin was placed in a PermeGear In-Line flow-through diffusion cell system (PermeGear; Hellertown, PA, USA) allowing horizontal flow underneath the diffusion cell skin area. PBS, pH 7.4 was used as the receiver solution. Skin samples on the diffusion apparatus were maintained at 32°C, typical skin surface temperature, using a circulating water bath. The diffusion experiments were initiated by charging the donor compartment with 0.25 mL of 60µg/ml GVNP suspension in PBS. Samples were collected from the receiver compartment at 4h-intervals for a total of 24 h. Immunological analysis. Five to 10 µL receiver solution samples were spotted onto 0.45 µm Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Boston, MA). Membranes were blocked in blocking buffer (PBS, with 0.01% Tween 20, 3% bovine serum albumin added) followed by incubation in blocking buffer containing GvpA antisera14 at a 1:1,000 dilution. Membranes were washed in washing buffer (PBS, with 0.01% Tween 20 added), incubated in blocking buffer supplemented with alkaline phosphatase labeled goat antirabbit secondary antibodies (1:2,500 dilution, Sigma Aldrich; St. Louis, MO, USA), and developed using the 1-Step NBT/BCIP substrate (Thermo Fisher Scientific; Rockford, IL, USA) according to the manufacturer's specifications. Membranes were photographed, and the intensity


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of spots quantified by densitometry using ImageJ software (http://imagej.nih.gov/ij/). Fluorescence microscopy. After completion of the 24 h diffusion, skin samples were collected and flash-frozen in optimal cutting temperature compound (Sakura Finetek USA Inc., Torrance, CA, USA) under liquid nitrogen. The skin was cryotomed and transverse skin slices were placed onto glass microscopy slides and kept frozen at -20°C until they were used for probing with rabbit anti-GvpA antisera. In brief, for the immunofluorescent staining procedure, frozen slides were thawed and rehydrated in PBS for 5 min. An Avidin blocking (USBiologicals, Santa Clara, CA) solution 0.1% (wt/vol) was applied for 15 min to block endogenous biotin. After 2 rinses with PBS for 5 min per wash, sections were blocked with PBS with 3% BSA (wt/vol) and 0.01% (vol/vol) Triton X-100 for 30 min (primary antibody diluent). Sections were then incubated overnight at 4°C with GvpA antibody solution in a solution of PBS with 3% BSA. The following day, sections were washed once with PBS with 0.1 % Triton-X100 for 10 min followed by 3 washes in PBS at 5 min per wash. Secondary antibody (goat-anti-rabbit-AlexaFlour-568, Invitrogen, Grand Island, NY) was prepared in secondary antibody diluent (PBS with 0.01% Triton X-100). Incubation with secondary antibody was performed for 1 h at room temperature. Sections were washed with 0.1% TritonX-100 for 10 min followed by 3 washes in PBS at 5 min per wash. DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) (Molecular Probes, Eugene, OR) was used to stain the DNA after which slides were mounted using VectaShield mounting media (Vector Laboratories, Burlingame, CA) and viewed at 20x and 40x magnification using a Nikon Laser confocal A1 microscope using previously described methods42. Images were acquired and processed using Nikon Elements software43,44. Cytotoxicity assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Cell Biolabs, San Diego, CA) was used to determine the effects of GVNPs on the cellular


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viability of HEK293 (Human embryonic kidney epithelial, ATCC CRL-1573) and H9c2(2-1) (Rat embryonic heart myoblasts, ATCC CRL-1446) cells. Briefly, cells were seeded into 96-well cell culture plates (Corning Costar, Tewksbury, MA) at an initial density of 7,500 cells/well, respectively, and cultured for 24 h. Cells were treated with a range of concentrations of GVNPs in PBS (62.5 - 500 µg/mL of GVNPs in PBS) and incubated for 24 h. The effect of GVNPs on cell viability was plotted as percent viability vs. concentration. Biological Materials. The Halobacterium sp. strain used for this study was NRC-1 (ATCC 700922/JCM11081)10 from the DasSarma Laboratory collection. Mammalian cell lines used were HEK-293 (ATCC CRL-1573) and H9c2(2-1) (ATCC CRL-1446), and were obtained from American Type Culture Collection (ATCC; Rockville, MD). FIGURES

1 Figure 1. Particle characterization. Mean diameter for GVNPs is presented with the polydispersity index (PDI). GVNP mean diameter = 245.08 ± 3.11 nm with a PDI of 0.12 ± 0.03 (n = 6).


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2 Figure 2. Schematic representation of flow-through diffusion apparatus. 1) MN treatment of Yucatan miniature pig skin using a 5 MN array 41, 2) Receiver solution (PBS) preparation for the diffusion flow-through and collection. Diffusion apparatus (PermeGear In-Line cell) clamped together once the MN treated skin has been gently placed inside. 3) Donor GVNP solution added. 4) Receiver solution collected and analyzed. Picture modified from Milewski et al.40

3 Figure 3. In-vitro permeation profile of GVNPs using GvpA protein immunoassay. A)


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Microneedle-treated and untreated skin were tested to show the amount of GVNPs permeated over every 4 h for a total of 24 h. Graph represents permeated amount of GVNPs measured over time. B) Represents cumulative permeation of GVNPs over 24 h after calculating the difference in permeation through untreated skin. 15 µg GVNPs were suspended in PBS. With MN treatment after 24 h = 0.22 ± 0.16 µg (n=3).

4 Figure 4. Fluorescent confocal microscopy. Yucatan miniature pig skin post diffusion 24-hour experiment. A) MN-treated skin negative control without added GVNPs. B) MN-treated skin with GVNPs added. Samples were stained with nuclear stain (DAPI, blue) and antisera for GvpA, followed by fluorescently-tagged (Alexa568, red) secondary anti-rabbit antibodies. Scale bars are 500 µm.


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H9c2

HEK293 100

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Concentration of NRC1-GVNPs (µg/ml)

5 Figure 5. Cytotoxicity of GVNPs testing using MTT-assay. HEK293 and H9c2(2-1) cells incubated with GVNPs for 24 h show close to 100% cell viability when tested with the MTT assay. Triton-X100 was used as a positive control to induce toxicity and showed 8% cell viability. Phosphate buffered saline (PBS, pH 7.4) was used as a negative control and cells showed 100% viability (represented as mean and standard deviation for n=3 sample sets).


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ACKNOWLEDGEMENTS The authors would like to thank Dr. G. Kao and her group at the Dermatopathology Laboratory Core facility, School of Medicine, Baltimore, for providing high quality microscopy images and frozen skin sections and Dr. M.R. Prausnitz’s Laboratory for Drug Delivery, Georgia Institute of Technology for providing microneedles. This work was supported by Bill & Melinda Gates Foundation grant OPP1061509 and National Institutes of Health grant AI107634 to S.D.


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Sremac, M.; Stuart, E. S. SIVsm Tat, Rev, and Nef1: Functional Characteristics of R-GV Internalization on Isotypes, Cytokines, and Intracellular Degradation. BMC Biotechnol. 2010, 10, 54.

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Childs, T. S.; Webley, W. C. In Vitro Assessment of Halobacterial Gas Vesicles as a Chlamydia Vaccine Display and Delivery System. Vaccine 2012, 30 (41), 5942–5948.

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Stuart, E. S.; Morshed, F.; Sremac, M.; DasSarma, S. Antigen Presentation Using Novel Particulate Organelles from Halophilic Archaea. J. Biotechnol. 2001, 88 (2), 119–128.

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Dutta, S.; DasSarma, P.; DasSarma, S.; Jarori, G. K. Immunogenicity and Protective Potential of a Plasmodium Spp. Enolase Peptide Displayed on Archaeal Gas Vesicle Nanoparticles. Malar. J. 2015, 14, 406.

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Balakrishnan, A.; DasSarma, P.; Bhattacharjee, O.; Kim, J. M.; DasSarma, S.; Chakravortty, D. Halobacterial Nano Vesicles Displaying Murine Bactericidal Permeability-Increasing Protein Rescue Mice from Lethal Endotoxic Shock. Sci. Rep. 2016, 6, 33679.

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DasSarma, P.; Karan, R.; Kim, J.-M.; Pecher, W.; DasSarma, S. Bioengineering Novel Floating Nanoparticles for Protein and Drug Delivery. Mater. Today Proc. 2016, 3 (2), 206–210.

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Pecher, W.; Kim, J.-M.; DasSarma, P.; Karan, R.; Sinnis, P.; DasSarma, S. Halobacterium Expression

System

for

Production

of

Full-Length

Plasmodium

Falciparum

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Babu, S.; Fan, C.; Stepanskiy, L.; Uitto, J.; Papazoglou, E. Effect of Size at the Nanoscale and Bilayer Rigidity on Skin Diffusion of Liposomes. J. Biomed. Mater. Res. - Part A 2009, 91 (1), 140–148.

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Passerini, N.; Gavini, E.; Albertini, B.; Rassu, G.; Di Sabatino, M.; Sanna, V.; Giunchedi, P.; Rodriguez, L. Evaluation of Solid Lipid Microparticles Produced by Spray Congealing for Topical Application of Econazole Nitrate. J. Pharm. Pharmacol. 2009, 61 (5), 559– 567.

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Schneider, M.; Stracke, F.; Hansen, S.; Schaefer, U. F. Nanoparticles and Their Interactions with the Dermal Barrier. Dermatoendocrinol. 2009, 1 (4), 197–206.

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Foldvari, M.; Badea, I.; Wettig, S.; Baboolal, D.; Kumar, P.; Creagh, A. L.; Haynes, C. A. Topical Delivery of Interferon Alpha by Biphasic Vesicles: Evidence for a Novel Nanopathway across the Stratum Corneum. Mol. Pharm. 2010, 7 (3), 751–762.

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Wermeling, D. P.; Banks, S. L.; Hudson, D. a; Gill, H. S.; Gupta, J.; Prausnitz, M. R.; Stinchcomb, A. L. Microneedles Permit Transdermal Delivery of a Skin-Impermeant Medication to Humans. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (6), 2058–2063.


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DasSarma, P.; Negi, V. D.; Balakrishnan, A.; Karan, R.; Barnes, S.; Ekulona, F.; Chakravortty, D.; DasSarma, S. Haloarchaeal Gas Vesicle Nanoparticles Displaying Salmonella SopB Antigen Reduce Bacterial Burden When Administered with Live Attenuated Bacteria. Vaccine 2014, 32 (35), 4543–4549.

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DasSarma, P.; Negi, V. D.; Balakrishnan, A.; Kim, J.-M.; Karan, R.; Chakravortty, D.; DasSarma, S. Haloarchaeal Gas Vesicle Nanoparticles Displaying Salmonella Antigens as a Novel Approach to Vaccine Development. Procedia Vaccinol. 2015, 9, 16–23.

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Sremac, M.; Stuart, E. S. Recombinant Gas Vesicles from Halobacterium Sp. Displaying SIV Peptides Demonstrate Biotechnology Potential as a Pathogen Peptide Delivery Vehicle. BMC Biotechnol. 2008, 8, 9.

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Stuart, E. S.; Morshed, F.; Sremac, M.; DasSarma, S. Cassette-Based Presentation of SIV Epitopes with Recombinant Gas Vesicles from Halophilic Archaea. J. Biotechnol. 2004, 114 (3), 225–237.

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Karan, R.; Capes, M. D.; DasSarma, P.; DasSarma, S. Cloning, Overexpression, Purification, and Characterization of a Polyextremophilic Beta-Galactosidase from the Antarctic Haloarchaeon Halorubrum Lacusprofundi. BMC Biotechnol. 2013, 13, 3.

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DasSarma, P.; Coker, J. A.; Huse, V.; DasSarma, S. Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology. In Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology; Flickinger, M., Ed.; Wiley, Johns & Sons, Inc, Hoboken, 2010; pp 2769–2777.

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Karan, R.; DasSarma, P.; Balcer-Kubiczek, E.; Weng, R. R.; Liao, C.-C.; Goodlett, D. R.; Ng, W. V.; Dassarma, S. Bioengineering Radioresistance by Overproduction of RPA, a Mammalian-Type Single-Stranded DNA-Binding Protein, in a Halophilic Archaeon. Appl.


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Milewski, M.; Paudel, K. S.; Brogden, N. K.; Ghosh, P.; Banks, S. L.; Hammell, D. C.; Stinchcomb, A. L. Microneedle-Assisted Percutaneous Delivery of Naltrexone Hydrochloride in Yucatan Minipig: In Vitro-in Vivo Correlation. Mol. Pharm. 2013, 10 (10), 3745–3757.

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Milewski, M.; Yerramreddy, T. R.; Ghosh, P.; Crooks, P. A.; Stinchcomb, A. L. In Vitro Permeation of a Pegylated Naltrexone Prodrug across Microneedle-Treated Skin. J. Control. Release 2010, 146 (1), 37–44.

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Heine, S. J.; Diaz-McNair, J.; Andar, A. U.; Drachenberg, C. B.; van de Verg, L.; Walker, R.; Picking, W. L.; Pasetti, M. F. Intradermal Delivery of Shigella IpaB and IpaD Type III Secretion Proteins: Kinetics of Cell Recruitment and Antigen Uptake, Mucosal and Systemic Immunity, and Protection across Serotypes. J. Immunol. 2014, 192 (4), 1630– 1640.

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Heine, S. J.; Diaz-McNair, J.; Andar, A. U.; Drachenberg, C. B.; Van De Verg, L.; Walker, R.; Picking, W. L.; Pasetti, M. F. Intradermal Delivery of Shigella IpaB and IpaD Type III Secretion Proteins: Kinetics of Cell Recruitment and Antigen Uptake, Mucosal and Systemic Immunity, and Protection across Serotypes. J. Immunol. 2014, 192 (4),


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1630–1640.


23

Halobacterium NRC-1

Buoyant GVNPs float

Molecular Pharmaceutics Page 24 of 25 up from cell debris

3 4 5 6 Skin 7

............

................................. .... ........... ........... ... . . . ..

1 2 Microneedles

.. ............ ........

..

Lyse cells and extract GVNPs


Collect permeated GVNPs

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable

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