Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

The sustained delivery of antisense oligodeoxynucleotides using biodegradable polymer microspheres

Antisense technology is a novel drug discovery method, which provides an essential tool for directly using gene sequence information to rationally design specific inhibitions of mRNA, to treat a wide range of diseases. The efficacy of naked oligodeoxynucleotides (ODNs) is relatively short lived due to rapid degradation in vivo. The entrapment of ODNs within biodegradable sustained-release delivery systems may improve ODN stability and reduce dose required for efficacy. Biodegradable polymer microspheres were evaluated as delivery devices for ODNs and ribozymes. Poly(lactide-co-glycolide) polymers were used due to their biocompatibility and non toxic degradation products. Microspheres were prepared using a double emulsion-deposition method and the formulations characterised. In vitro release profiles were characterised by an initial burst effect during the first 48 hours of release followed by a more sustained release. The release profiles were influenced by microsphere size, copolymer molecular weight, copolymer ratio, ODN loading, ODN length, and ODN chemistry. The serum stability of ODNs was significantly improved when entrapped within polymer microspheres. The cellular association of ODNs entrapped within small spheres (1-2μm) was improved by approximately 20-fold in A431 carcinoma cells compared with free ODNs. Fluorescence microscopy studies showed a more diffuse subcellular distribution when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. For in vivo evaluation, polymer microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain. Free ODN resulted in a punctate cellular distribution after 24 hours. In comparison ODN delivered using polymer microspheres were intensely visible in cells 48 hours post administration, and fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Whole-body autoradiography was also used to evaluate the biodistribution of free tritium labelled ODN and ODN entrapped microspheres, following subcutaneous administration to Balb-C mice. Polymer entrapped ODN gave a similar biodistribution to free ODN. Free ODN was distributed within 24 hours, whereas polymer released ODN was observed still presented in organs and at the site of administration seven days post administration.

Incorporation and release of macromolecules from biodegradable polymer vehicles

The initial objective of this work was to evaluate and introduce fabrication techniques based on W/0/W double emulsion and 0/W single emulsion systems with solvent evaporation for the incorporation of a surrogate macromolecule (BSA) into microspheres and microcapsules fabricated using P(HB-HV}, PEA and their blends. Biodegradation, expressed as changes in the gross and ultrastructural morphology of BSA loaded microparticulates with time was monitored using SEM concomitant with BSA release. Spherical microparticulates were successfully fabricated using both the W/0/W and 0/W emulsion systems. Both microspheres and microcapsules released BSA over a period of 24 to 26 days. BSA release from P(HB-HV)20% PCL 11 microcapsules increased steadily with time, while BSA release from all other microparticulates was characterised by an initial lag phase followed by exponential release lasting 6-11 days. Microcapsules were found to biodegrade more rapidly than microspheres fabricated from the same polymer. The incubation of microparticulates in newborn calf serum; synthetic gastric juice and pancreatin solution showed that microspheres and microcapsules were susceptible to enzymatic biodegradation. The in vitro incubation of microparticulates in Hank's buffer demonstrated limited biodegradation of microspheres and microcapsules by simple chemical hydrolysis. BSA release was thought to ocurr as a result of the macromolecule diffusing through either inherent micropores or via pores and channels generated in situ by previously dissolved BSA. However, in all cases, irrespective of percentage loading or fabrication polymer, low encapsulation efficiencies were obtained with W/0/W and 0/W techniques (4.2±0.9%- 15.5±0.5%,n=3), thus restricting the use of these techniques for the generation of microparticulate sustained drug delivery devices. In order to overcome this low encapsulation efficiency, a W/0 single emulsion technique was developed and evaluated in an attempt to minimise the loss of the macromolecule into the continuous aqueous phase and increase encapsulation efficiency. Poly(lactide-co-glycolide) [PLCG] 75:25 and 50:50, PEA alone and PEA blended with PLCG 50:50 to accelerate biodegradation, were used to microencapsulate the water soluble antibiotic vancomycin, a putative replacement for gentamicin in the control of bacterial infection in orthopaedic surgery especially during total hip replacement. Spherical microspheres (17.39±6.89~m,n=74-56.5±13.8~m,n=70) were successfully fabricated with vancomycin loadings of 10, 25 and 50%, regardless of the polymer blend used. All microspheres remained structurally intact over the period of vancomycin release and exhibited high percentage yields( 40. 75±2 .86%- 97.16±4.3%,n=3)and encapsulation efficiencies (47.75±9.0%- 96.74±13.2%,n=12). PLCG 75:25 microspheres with a vancomycin loading of 50% were judged to be the most useful since they had an encapsulation efficiency of 96.74+13.2%, n=12 and sustained therapeutically significant vancomycin release (15-25μg/ml) for up to 26 days. This work has provided the means for the fabrication of a spectrum of prototype biodegradable microparticulates, whose biodegradation has been characterised in physiological media and which have the potential for the sustained delivery of therapeutically useful macromolecules including water soluble antibiotics for orthopaedic applications.

Controlled release in inhalation

Increasingly complicated medication regimens associated with the necessity of the repeated dosing of multiple agents used in treating pulmonary disease has been shown to compromise both disease management and patient convenience. In this study the viability of spray drying to introduce controlled release vectors into dry powders for inhalation was investigated. The first experimental section highlights the use of leucine in producing highly respirable spray dried powders, with in vitro respirable fractions (Fine particle fraction, FPF: F < 5µm) exceeding 80% of the total dose. The second experimental chapter introduces the biocompatible polymer chitosan (mw 190 – 310 kDa) to formulations containing leucine with findings of increased FPF with increasing leucine concentration (up to 82%) and the prolonged release of the active markers terbulataline sulfate (up to 2 hours) and beclometasone dipropionate (BDP: up to 12 hours) with increasing chitosan molecular weight. Next, the thesis details the use of a double emulsion format in delivering the active markers salbutamol sulfate and BDP at differing rates; using the polymers poly-lactide co-glycolide (PLGA 50:50 and PLGA 75:25) and/or chitosan incorporating leucine as an aerosolisation enhancer the duration of in vitro release of both agents reaching 19 days with FPF exceeding 60%. The final experimental chapter involves dual aqueous and organic closed loop spray drying to create controlled release dry powders for inhalation with in vitro sustained release exceeding 28 days and FPF surpassing 55% of total loaded dose. In conclusion, potentially highly respirable sustained release dry powders for inhalation have been produced by this research using the polymers chitosan and/or PLGA as drug release modifiers and leucine as an aerosolisation enhancer.

Formulation and characterisation of an effective particulate delivery vehicle for the novel sub-unit vaccine antigen, Ag85B-ESAT-6

This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Further, freeze-drying studies were carried out with both sugar-based and amino acid-based cryoprotectants, in order to formulate a stable freexe-dried product. Finally, environmental scanning electron microscopy (ESEM) was investigated as a potential alternative to conventional SEM for the morphological investigation of microsphere formulations. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-ϒ), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.

Evaluation of biodegradable polymer microspheres and an anionic dendrimer for the potential delivery of antisense oligonucleotides

Antisense oligonucleotides (AODNs) can selectively inhibit individual gene expression by binding specifically to rnRNA. The over-expression of the epidermal growth factor receptor (EGFR) has been observed in human breast and glioblastoma tumours and therefore AODNs designed to target the EGFR would be a logical approach to treat such tumours. However, poor pharmacokinetic/pharmacodynamic and cellular uptake properties of AODNs have limited their potential to become successful therapeutic agents. Biodegradable polymeric poly (lactide-co-glycolide) (P(LA-GA)) and dendrimer delivery systems may allow us to overcome these problems. The use of combination therapy of AODNs and cytotoxic agents such as 5-fluorouracil (5-FU) in biodegradable polymeric formulations may further improve therapeutic efficacy. AODN and 5-FU were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations (double emulsion method) and release profiles determined in vitro. The release rates (biphasic) of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Sustained release over 35 days was observed in both types of formulation. Naked and microsphere-loaded AODN and 5-FU (in separate formulations) were tested on an A431 vulval carcinoma cell line. Combining naked or encapsulated drugs produced a greater reduction in viable cell number as compared with either agent alone. However, controls and Western blotting indicated that non-sequence specific cytotoxic effects were responsible for the differences in viable cell number. The uptake properties of an anionic dendrimer based on a pentaerythritol structure covalently linked to AODNs (targeting the EGFR) have been characterised. The cellular uptake of AODN linked to the dendrimer was up to 3.5-fold higher in A431 cells as compared to naked AODN. Mechanistic studies suggested that receptor-mediated and adsorptive (binding protein-mediated) endocytosis were the predominant uptake mechanisms for the dendrimer-AODN. RNase H cleavage assay suggested that the dendrimer-AODN was able to bind and cleave the target site. A reduction of 20%, 28% and 45% in EGFR expression was observed with 0.05μM, 0.1μM and 0.5μM dendrimer-AODN treatments respectively with a reduction in viable cell number. These results indicated that the dendrimer delivery system may reduce viable cell number by an antisense specific mechanism.

Novel formulations for antigen delivery using biodegradable polymers: new approaches for the use of new and established adjuvants

The use of immunological adjuvants has been established since 1924 and ever since many candidates have been extensively researched in vaccine development. The controlled release of vaccine is another area of biotechnology research, which is advancing rapidly with great potential and success. Encapsulation of peptide and protein drugs within biodegradable microspheres has been amongst the most successful of approaches within the past decade. The present studies have focused on combining the advantages of microsphere delivery systems composed of biodegradable polylactide (PLLA) and polylactide-co-glycolide (PLGA) polymers with that of safe and effective adjuvants. The research efforts were directed to the development of single-dose delivery vehicles which, can be manufactured easily, safely, under mild and favourable conditions to the encapsulated antigens. In pursuing this objective non ionic block copolymers (NIBCs) (Pluronics@ LI01 and L121) were incorporated within poly-dl-lactide (PDLA) micorospheres prepared with emulsification-diffusion method. LI0I and L121 served both as adjuvants and stabilising agents within these vaccine delivery vehicles. These formulations encapsulating the model antigens lysozyme, ovalbumin (OVA) and diphtheria toxoid (DT) resulted in high entrapment efficiency (99%), yield (96.7%) and elicited high and sustained immune response (IgG titres up to 9427) after one single administration over nine months. The structural integrity of the antigens was preserved within these formulations. In evaluating new approaches for the use of well-established adjuvants such as alum, these particles were incorporated within PLLA and PLGA microspheres at much lesser quantities (5-10 times lower) than those contained within conventional alum-adsorbed vaccines. These studies focused on the incorporation of the clinically relevant tetanus toxoid (TT) antigen within biodegradable microspheres. The encapsulation of both alum particles and TT antigen within these micropheres resulted in preparations with high encapsulation efficiency (95%) and yield (91.2%). The immune response to these particles was also investigated to evaluate the secretion of serum IgG, IgG1, IgG2a and IgG2b after a single administration of these vaccines. The Splenic cells proliferation was also investigated as an indication for the induction of cell mediated immunity. These particles resulted in high and sustained immune response over a period of 14 months. The stability of TT within particles was also investigated under dry storage over a period of several months. NIBC microspheres were also investigated as potential DNA vaccine delivery systems using hepatitis B plasmid. These particles resulted in micro spheres of 3-5 μm diameter and were shown to preserve the integrity of the encapsulated (27.7% entrapment efficiency) hepatitis B plasmid.

Particulate delivery systems for vaccines

This review focuses on the use of particulate delivery systems for the purposes of immunization. This includes poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, chitosan, and other biodegradable polymers. These systems are evaluated in terms of their use as carriers for protein subunit and DNA vaccines. There is an extensive focus on recent literature, the understanding of biological interactions, and relation of this to our present understanding of immunological mechanisms of action. In addition, there is consideration of formulation techniques including emulsification, solvent diffusion, DNA complexation, and entrapment. The diversity of formulation strategies presented is a testament to the exponential growth and interest in the area of vaccine delivery systems. A case study for the application of particulate vaccine carriers is assessed in terms of vaccine development and recent insights into the possible design and application of vaccines against two of the most important pathogens that threaten mankind and for which there is a significant need: Mycobacterium tuberculosis and human immunodeficiency virus. This review addresses the rationale for the use of particulate delivery systems in vaccine design in the context of the diversity of carriers for DNA- and protein-based vaccines and their potential for application in terms of the critical need for effective vaccines. © 2005 by Begell House, Inc.

Developing solid particulate vaccine adjuvants - surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Developing solid particulate vaccine adjuvants:surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Through-thickness plasma modification of biodegradable and nonbiodegradable porous polymer constructs

Pure poly(lactide-co-glycolide) and polystyrene surfaces are not very suitable to support cell adhesion/ spreading owing to their hydrophobic nature and low surface energy. The interior surfaces of large porous 3D scaffolds were modified and activated using radio-frequency, low-pressure air plasma. An increase in the wettability of the surface was observed after exposure to air plasma, as indicated by the decrease in the contact angles of the wet porous system. The surface composition of the plasma-treated polymers was studied using X-ray photoelectron spectroscopy. pH-dependent zeta-potential measurements confirm the presence of an increased number of functional groups. However, the plasma-treated surfaces have a less acidic character than the original polymer surfaces as seen by a shift in their isoelectric point. Zeta-potential, as well as contact angle measurements, on 3D scaffolds confirm that plasma treatment is a useful tool to modify the surface properties throughout the interior of large scaffolds. © 2008 Wiley Periodicals, Inc.

Poly(glycerol adipate-co-ω-pentadecalactone) spray-dried microparticles as sustained release carriers for pulmonary delivery

Purpose: The aim of this work was to optimize biodegradable polyester poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, microparticles as sustained release (SR) carriers for pulmonary drug delivery. Methods: Microparticles were produced by spray drying directly from double emulsion with and without dispersibility enhancers (L-arginine and L-leucine) (0.5-1.5%w/w) using sodium fluorescein (SF) as a model hydrophilic drug. Results: Spray-dried microparticles without dispersibility enhancers exhibited aggregated powders leading to low fine particle fraction (%FPF) (28.79±3.24), fine particle dose (FPD) (14.42±1.57 μg), with a mass median aerodynamic diameter (MMAD) 2.86±0.24 μm. However, L-leucine was significantly superior in enhancing the aerosolization performance ( L-arginine:%FPF 27.61±4.49-26.57±1.85; FPD 12.40±0.99-19.54±0.16 μg and MMAD 2.18±0.35-2. 98±0.25 μm, L-leucine:%FPF 36.90±3.6-43.38±5. 6; FPD 18.66±2.90-21.58±2.46 μg and MMAD 2.55±0.03-3. 68±0.12 μm). Incorporating L-leucine (1.5%w/w) reduced the burst release (24.04±3.87%) of SF compared to unmodified formulations (41.87±2.46%), with both undergoing a square root of time (Higuchi's pattern) dependent release. Comparing the toxicity profiles of PGA-co-PDL with L-leucine (1.5%w/w) (5 mg/ml) and poly(lactide-co-glycolide), (5 mg/ml) spray-dried microparticles in human bronchial epithelial 16HBE14o-cell lines, resulted in cell viability of 85.57±5.44 and 60.66±6.75%, respectively, after 72 h treatment. Conclusion:The above data suggest that PGA-co-PDL may be a useful polymer for preparing SR microparticle carriers, together with dispersibility enhancers, for pulmonary delivery. © Springer Science+Business Media, LLC 2011.

Bioengineered Approaches to Prevent Hypertrophic Scar Contraction

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

Cationic Bovine Serum Albumin (CBA) conjugated Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles for extended delivery of methotrexate into brain tumor

Current treatment strategies for the treatment of brain tumor have been hindered primarily by the presence of highly lipophilic insurmountable blood-brain barrier (BBB). The purpose of current research was to investigate the efficiency of engineered biocompatible polymeric nanoparticles (NPs) as drug delivery vehicle to bypass the BBB and enhance biopharmaceutical attributes of anti-metabolite methotrexate (MTX) encapsulated NPs. The NPs were prepared by solvent diffusion method using cationic bovine serum albumin (CBA), and characterized for physicochemical parameters such as particle size, polydispersity index, and zeta-potential; while the surface modification was confirmed by FTIR, and NMR spectroscopy. Developed NPs exhibited zestful relocation of FITC tagged NPs across BBB in albino rats. Further, hemolytic studies confirmed them to be non-toxic and biocompatible as compared to free MTX. In vitro cytotoxicity assay of our engineered NPs on HNGC1 tumor cells proved superior uptake in tumor cells; and elicited potent cytotoxic effect as compared to plain NPs and free MTX solution. The outcomes of the study evidently indicate the prospective of CBA conjugated poly (D,L-lactide-co-glycolide) (PLGA) NPs loaded with MTX in brain cancer bomber with amplified capability to circumvent BBB.

Investigation of properties affecting controlled release of macromolecules from PLGA microspheres

Poly(lactide-co-glycolide), or PLGA, microspheres offer a widely-studied biodegradable option for controlled release of therapeutics. An array of fabrication methodologies have been developed to produce these microspheres with the capacity to encapsulate therapeutics of various types; and produce microspheres of a wide range of sizes for different methods of delivery. The encapsulation, stability, and release profiles of therapeutic release based on physical and thermodynamic properties has also been studied and modeled to an extent. Much research has been devoted to tailoring formulations for improved therapeutic encapsulation and stability as well as selective release profiles. Despite the breadth of available research on PLGA microspheres, further analysis of fundamental principles regarding the microsphere degradation, formation, and therapeutic encapsulation is necessary. This work aims to examine additional fundamental principles related to PLGA microsphere formation and degradation from solvent-evaporation of preformed polymer. In particular, mapping the development of the acidic microenvironment inside the microsphere during degradation and erosion is discussed. Also, the effect of macromolecule size and conformation is examined with respect to microsphere diameter and PLGA molecular weight. Lastly, the effects of mechanical shearing and protein exposure to aqueous media during microsphere formation are examined. In an effort to better understand the acidic microenvironment development across the microsphere diameter, pH sensitive dye conjugated to protein that undergoes conformational change at different acidic pH values was encapsulated in PLGA microspheres of diameters ranging from 40 µm to 80 µm, and used in conjunction with fluorescence resonance energy transfer to measure the radial pH change in the microspheres. Qualitative analysis of confocal micrographs was used to correlate fluorescence intensity with pH value, and obtain the radial pH across the center of the microsphere. Therapeutic encapsulation and release from polymeric microspheres is governed by an interconnected variety of factors, including the therapeutic itself. The globular protein bovine serum albumin, and the elongated and significantly smaller enzyme, lysozyme, were encapsulated in PLGA microspheres ranging from 40 µm to 80 µm in diameter. The initial surface morphology upon microsphere formation, release profiles, and microsphere erosion characteristics were explored in an effort to better understand the effect of protein size, conformation, and known PLGA interaction on the formation and degradation of PLGA microspheres and macromolecule release, with respect to PLGA molecular weight and microsphere diameter. In addition to PLGA behavior and macromolecule behavior, the effect of mechanical stresses during fabrication was examined. Two similar solvent extraction techniques were compared for the fabrication of albumin loaded microspheres. In particular, the homogeneity of the microspheres as well as capacity to retain encapsulated albumin were compared. This preliminary study paves the way for a more rigorous treatment of the effect of mechanical forces present in popular microsphere fabrication. Several factors affecting protein release from PLGA microspheres are examined herein. The technique explored for spatial resolution of the pH inside the microsphere proved mildly effective in producing a reliable method of mapping microsphere pH changes. However, notable trends with respect to microsphere size, PLGA molecular weight, and microsphere porosity were observed. Proposed methods of improving spatial resolution of the acidic microenvironment are also provided. With respect to microsphere formation, studies showed that albumin and lysozyme had little effect on the internal homogeneity of the microsphere. Rather, ionic interactions with PLGA played a more significant role in the encapsulation and release of each macromolecule. Studies also showed that higher instances of mechanical stress led to less homogeneous microspheres with lower protein encapsulation. This suggests that perhaps instead of or in addition to modifying the microsphere formation formulation, the fabrication technique itself should be more closely considered in achieving homogeneous microspheres with desired loading.