V. Prasad Shastri

Novel Microemulsion Enhancer Formulation for Simultaneous Transdermal Delivery of Hydrophilic and Hydrophobic Drugs

AbstractPurpose. Microemulsion (ME) systems allow for the microscopic co-incorporation of aqueous and organic phase liquids. In this study, the phase diagrams of four novel ME systems were characterized.nMethods. Water and IPM composed the aqueous and organic phases respectively, whereas Tween 80 served as a nonionic surfactant. Transdermal enhancers such as n-methyl pyrrolidone (NMP) and oleyl alcohol were incorporated into all systems without disruption of the stable emulsion.nResults. A comparison of a W/O ME with an O/W ME of the same system for lidocaine delivery indicated that the O/W ME provides significantly greater flux (p < 0.025). The water phase was found to be a crucial component for flux of hydrophobic drugs (lidocaine free base, estradiol) as well as hydrophilic drugs (lidocaine HCl, diltiazem HCl). Furthermore, the simultaneous delivery of both a hydrophilic drug and a hydrophobic drug from the ME system is indistinguishable from either drug alone. Enhancement of drug permeability from the O/W ME system was 17-fold for lidocaine free base, 30-fold for lidocaine HCl, 58-fold for estradiol, and 520-fold for diltiazem HCl.nConclusions. The novel microemulsion systems in this study potentially offers many beneficial characteristics for transdermal drug delivery.

The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function.

Chemical and morphological characteristics of a biomaterial surface are thought to play an important role in determining cellular differentiation and apoptosis. In this report, we investigate the effect of nanoparticle (NP) assemblies arranged on a flat substrate on cytoskeletal organization, proliferation and metabolic activity on two cell types, Bovine aortic endothelial cells (BAECs) and mouse calvarial preosteoblasts (MC3T3-E1). To vary roughness without altering chemistry, glass substrates were coated with monodispersed silica nanoparticles of 50, 100 and 300 nm in diameter. The impact of surface roughness at the nanoscale on cell morphology was studied by quantifying cell spreading, shape, cytoskeletal F-actin alignment, and recruitment of focal adhesion complexes (FAC) using image analysis. Metabolic activity was followed using a thiazolyl blue tetrazolium bromide assay. In the two cell types tested, surface roughness introduced by nanoparticles had cell type specific effects on cell morphology and metabolism. While BAEC on NP-modified substrates exhibited smaller cell areas and fewer focal adhesion complexes compared to BAEC grown on glass, MC3T3-E1 cells in contrast exhibited larger cell areas on NP-modified surfaces and an increased number of FACs, in comparison to unmodified glass. However, both cell types on 50 nm NP had the highest proliferation rates (comparable to glass control) whereas cells grown on 300 nm NP exhibited inhibited proliferation. Interestingly, for both cell types surface roughness promoted the formation of long, thick F-actin fibers, which aligned with the long axis of each cell. These findings are consistent with our earlier result that osteogenic differentiation of human mesenchymal progenitor cells is enhanced on NP-modified surfaces. Our finding that nanoroughness, as imparted by nanoparticle assemblies, effects cellular processes in a cell specific manner, can have far reaching consequences on the development of smart biomaterials especially for directing stem cell differentiation.

Non-degradable biocompatible polymers in medicine: past, present and future.

Polymers have a long history in medicine. Their uses to date range from traditional applications such as catheters, syringes, blood contacting extra corporeal devices to matrices for drug delivery, cell encapsulation and tissue regeneration. Polymers can be broadly classified on the basis of the reactivity of their chemical backbone (or susceptibility of the backbone to breakdown upon exposure to water, i.e., hydrolysis) as non-degradable and degradable. In this review, the polymers that exhibit no to very low degradation in aqueous and biological environments will be covered. The applications of various polymers both in traditional and emerging medical areas is discussed in the context of its chemical structure to better enable material selection for biomedical research.

Photocrosslinked anhydride systems for long-term protein release

Injectable delivery systems are attractive as vehicles for localized delivery of therapeutics especially in the context of regenerative medicine. In this study, the potential of photocrosslinked polyanhydride (PA) networks as an encapsulation matrix for long-term delivery of macromolecules was studied. The in vitro release of two model proteins (horseradish peroxidase (HRP) and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA)) was evaluated from crosslinked networks composed of sebacic acid dimethacrylate (MSA), 1,6-bis-carboxyphenoxyhexane dimethacrylate (MCPH), and poly(ethylene glycol) diacrylate (PEGDA), supplemented with calcium carbonate. Prior to incorporation into the networks, proteins were formulated by dilution in a cyclodextrin excipient followed by gelatin-based wet granulation. Protein release was quantified by activity assay (HRP) or fluorescence (FITC-BSA). Each protein was readily released from the networks with a unique release behavior. Most importantly, release of protein with retention of activity was achieved for durations ranging from 1 week to 4 months. The released HRP was additionally visualized using SDS-PAGE. In general, a more hydrophobic network resulted in slower rates of protein release. Incorporation of PEGDA into the matrices was critical for maintenance of integrity during degradation. These results suggest that this system may be useful as an injectable delivery system for long-term delivery of macromolecules.

Engineering a Material Surface for Drug Delivery and Imaging using Layer-by-Layer Assembly of Functionalized Nanoparticles

[*] Prof. V. P. Shastri, T. Soike, A. K. Streff, C. Guan, R. Ortega, M. Tantawy, Dr. C. Pino Institute for Macromolecular Chemistry University of Freiburg Stefan-Meier Strasse 31, 79104 Freiburg (Germany) E-mail: prasad.shastri@bioss.uni-freiburg.de; prasad.shastri@makro.uni-freiburg.de Prof. V. P. Shastri, T. Soike, A. K. Streff, C. M. Guan Department of Biomedical Engineering Vanderbilt University Nashville, TN 37232 (USA)

Modulation of protein release from photocrosslinked networks by gelatin microparticles.

Injectable delivery systems are attractive as vehicles for localized delivery of therapeutics especially in the context of regenerative medicine. In this study, photocrosslinked polyanhydride (PA) networks were modified by incorporation of microparticles to modulate long-term delivery of macromolecules. The in vitro release of two model proteins (horseradish peroxidase (HRP) and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA)) were evaluated from networks composed of sebacic acid dimethacrylate (MSA), 1,6-bis-carboxyphenoxyhexane dimethacrylate (MCPH), poly(ethylene glycol) diacrylate (PEGDA), and calcium carbonate (CaCO3), supplemented with gelatin microparticles or sodium chloride crystals. Prior to incorporation into the networks, proteins were formulated into granules by dilution with a cyclodextrin excipient and gelatin-based wet-granulation. Protein release was modulated by incorporation of microparticles into photocrosslinked PA networks, presumably by enabling aqueous channels through the matrix. Furthermore, a dual release system has been demonstrated by incorporation of protein in both the PA matrix and the gelatin microparticles. These results suggest that microparticle incorporation into the photocrosslinked PA system may be a useful strategy to modulate protein release in injectable delivery systems for the long-term delivery of macromolecules. These composites present an interesting class of materials for bone regeneration applications.

Influence of surface charge and protein intermediary layer on the formation of biomimetic calcium phosphate on silica nanoparticles

The biomineralization process in bone involves the assembly of calcium phosphate (CP) as a bio-inorganic nanocomposite in association with extracellular matrix proteins. Towards developing synthetic systems that can promote the formation of biomimetic hydroxyapatite, the formation of CP from solution onto the surfaces of a model system composed of silica nanoparticles (SNPs) of differing physiochemical characteristics was studied. In addition to varying charge, two protein mediators collagen and phosvitin were adsorbed onto the surface of SNPs to form protein-coated SNPs, and the precipitation of CP onto the SNP from solutions containing physiologically relevant concentrations of calcium and phosphate ions was studied. A comparison of zeta potential (ζ) versus pH isotherms between the protein-coated SNP and native protein reveals deviation in ζ that can be attributed to perturbation in the protein structure. Analysis of the ζ versus pH isotherms for CP–protein–silica nanocomposites and the native CP indicates that CP deposition on SNPs occurs in a heterogeneous manner with segregated regions of CP. Interestingly, SNP composites containing phosvitin exhibit lower component segregation relative to composites containing collagen, and that the difference in segregation originates from the difference in ζ of each adsorbed protein. This suggests that protein-mediated biomineralization might be governed by surface energetics as much as biology.

Non-covalent surface engineering of an alloplastic polymeric bone graft material for controlled protein release

Alloplastic materials, derived from poly(methylmethacrylate), such Bioplant-HTR, are a promising alternative to autologous bone in implant-dentistry and maxillofacial reconstruction. The clinical utility and outcomes using alloplasts such as HTR can be enhanced through the incorporation and release of proteins and growth factors. A simple, water-based process to surface engineer alloplast material to bear proteins has been developed. In this non-covalent process, the protein of choice is formulated into granules using gelatin-wet granulation and immobilized on the HTR alloplast surface, using water-soluble polymeric binders such as poly(vinyl alcohol) and Pluronics. The utility of this process has been verified using bovine serum albumin and horseradish peroxidase as model proteins. The process is capable of rendering these proteins on HTR surface in a reproducible manner, with formulated protein:HTR ratios less than 1:1 favoring more uniform surface coatings. By varying the ratio of the granulated protein to the HTR, surface protein concentration as high as 30 mug/mg of HTR particle can be achieved. By incorporating the protein-modified HTR particles with photocurable polymeric matrices and varying its hydrophobicity, sustained release of active HRP for at least 30 days was observed, with cumulative release ranging from 7-35% of loaded protein, depending on the protein:HTR ratio and the polymeric binder. The integrity of the released protein was also verified using SDS-PAGE gel and enzymatic assay. The simplicity of the surface modification strategy may make this suitable for ceramic and metal substrates as well.

Hydraulic Elevation of the Periosteum: A Novel Technique for Periosteal Harvest

Periosteum has been promoted as a potential substrate for tissue engineering. Its principal virtues are that it has a source of pluripotential mesenchymal cells and chondrogenic growth factors located in the cambium layer, and it can serve as a template for directional evolution of neo-tissue. The clinical use and in vitro study of periosteum-derived neo-tissue has been limited by the level of surgical skill required for harvest. Precise surgical technique, task-specific experience, adequate volume of procedures, and general surgical expertise are required for optimal harvest using the traditional periosteal elevator method. This report describes an easily mastered technique that preserves viability while providing the harvest of relatively large amounts of periosteum. Skeletally mature New Zealand white rabbits (11 males/20 tibias; 4 females/8 tibias; approximate weight 3.5 kg) and one Yucatan miniature pig were used for harvest of periosteum from the tibia using the traditional periosteal elevator and the developed hydraulic elevation approach. Histologic examination of the periosteal explants obtained by the developed method showed preservation of the cambium layer containing the progenitor cells necessary for the generation of neo-cartilage. This technique provides a simple method of harvesting large segments (>5 cm × 1 cm) of periosteum in a single procedure and may facilitate better exploitation of periosteum in tissue engineering.

Investigation of the transdermal transport of charged local anesthetics in the presence of triterpene saponin glycosides.

Percutaneous absorption and transdermal delivery of water-soluble drugs have proven to be challenging due to their low permeability through skin. Avicins which are triterpene saponin glycosides (TSGs) derived from the desert plant Acacia victoriae have not been investigated to date as chemical penetration enhancers due to their higher molecular weight (MW 2,000xa0Da). It was recently shown that avicins exhibit remarkable mobility across skin lipids in spite of their large size due to their unique chemical structure. In this study, the permeation of local anesthetics, lidocaine–HCl, prilocaine–HCl, and bupivacaine–HCL from aqueous vehicle, across full-thickness porcine skin was investigated in the presence of F094—a mixture of avicins. F094 was capable of enhancing the permeability of all three anesthetics from aqueous formulations at extremely low concentrations ranging from 0.1 to 1xa0% w/v. The enhancement, which ranged from 2- to 5-fold, was surprisingly independent of molecular weight of the anesthetics and showed clear correlation with aqueous phase solubility of the anesthetics. Since F094 was found to have no impact on the octanol/water partition coefficients of the anesthetics, this suggests that TSGs like avicins most likely impact the aqueous pathways (pericellular/pores within lipids) and as such represent an alternative means of enhancing the transdermal transport of charged drugs from water-based formulations.