Injectable hydrogels are important for use in tissue engineering due to their permeability and biocompatibility. Those that have shear thinning properties allow for minimally-invasive surgical procedures and a way to administer bioactive agents, and therapeutic cells by injection. Currently available injectable hydrogels have a single or dual input/stimulus for crosslinking which limits the range of mechanical properties and often utilize potentially toxic ultraviolet radiation that reduces viability of injected cells. To overcome these shortcomings, a tri-stimuli-responsive alginate-based injectable hydrogel was developed based on: 1) supramolecular complex formation between β-cyclodextrin (β-CD) conjugated alginate and thermo-responsive tri-block Pluronic® copolymers, 2) visible light crosslinking via acrylate conjugation, and 3) ionic crosslinking of the alginate backbone via exposure to calcium chloride. The capabilities of the novel multi-stimuli injectable hydrogel were demonstrated with a custom microfluidic devices (MFDs) to create microspheres encapsulating human mesenchymal stem cell (MSCs). These experiments proved that the new hydrogel was capable of serving as a stimuli responsive material for MSC cell delivery in the therapeutic range of 10-1000 µm in diameter. In order to enhance the drug delivery capabilities of the hydrogel, heparin sodium was conjugated onto the alginate backbone. The affinity of the growth factor, vascular endothelial growth factor (VEGF), to the heparin helped to prevent denaturing of the protein and improved vascularization. This new tri-crosslinking hydrogel with conjugated heparin allows the end-user to control the final physicomechanical and biochemical properties of the hydrogel using different external stimuli. The tri-crosslinking hydrogel is a versatile material that has great promise for a variety of soft tissue repair applications.

Many highly effective drugs display serious side-effects. To limit them, one can contain the drug in tiny containers, which are subsequently delivered toward targets inside the body. The entrapment of drug molecules prevents them from coming in contact with and thus causing damage to normal cells. Inherently, it is difficult to reach 100% efficiency of drug trapping and release when employing physical caps to seal the vehicles. Instead, we propose drug trapping inside the nanopores of hydrophobic silica particles by "hydrophobic trapping". This phenomenon is associated with the repulsive "force field" generated inside nanometer-sized hydrophobic channels that completely prevents aqueous solutions from entering the channels. We demonstrate the excellent trapping efficiency using C18-modified silica particles with 10 nm pores and the anticancer drug doxorubicin. The major challenge in using hydrophobic particles in biological applications is their tendency to cluster in aqueous media. To overcome it, we use surfactants as solubilization means. We have developed protocols that effectively solubilize the outer surface of the particles while preventing surfactant micelles from entering nanopores. Consequently, particles become well-dispersible in aqueous solutions, with the pre-loaded drug safely contained inside nanopores. Nanomaterials exhibit heterogeneity on their surfaces that impact their functional applications. Although techniques such as atomic force microscopy are great tools for studying nanomaterials with their excellent spatial resolution, they cannot probe the inner surface of porous structures. We have established a method of single-molecule ratiometric imaging that is currently the only technique able to provide the nanopolarity of adsorption sites located on the pore surface. We analyze the polarity distribution of adsorptions events for the solvatochromic probe Nile Red at the C18/acetonitrile interface and discover at least two different populations of adsorption sites. One of them corresponds to the polarity of surface silanol groups while the other sites have a polarity consistent with the environment inside the C18 organic layer. We also discover an additional adsorption mode situated at a polarity higher than exposed silanol surface that could presumably be linked to a different ionization state of the silanol groups. We are developing a method for resolving spectra of complex samples using two-dimensional hetero-correlation spectroscopy by correlating the intensity fluctuations in optical spectra to those of completely separated peaks in analytical separations. We demonstrate this methodology for fluorescence spectra and electrophoregrams of mixtures anthracene-pyrene. All the individual vibronic features that overlap in mixtures are cleanly extracted in cross-sections of the two-dimensional asynchronous spectrum.

The goal of this project was to develop a series of nano platforms for single cell analysis and drug delivery. Nanoparticles are a promising option to improve our medical therapies by controlling biodistribution and pharmacokinetics of therapeutics. Nanosystems also offer significant opportunity to improve current imaging modalities. The systems developed during this thesis work can be foundations for developing advanced therapies for obesity and improving our fundamental understandings of single cell behavior. The first of the two systems we attempt to create was a drug delivery system that could selectively target adipose tissue to deliver uncoupling agents and drive browning of adipose tissue and associated weight loss. Protonophores have a history of significant toxic side effects in cardiac and neuronal tissues a recently discovered protonophore, but BAM-15, has been shown to have reduced cytotoxicity. We hypothesized that the altered biodistribution of BAM-15 encapsulated in a nanoparticle could provide systemic weight loss with minimized side effects. The second system developed utilized quantum dots to create a fluorescent barcode that could be repeatedly identified using quantitative fluorescent emission readings. This platform would allow for the tracking of individual cells, allowing repeat interrogation across time and space in complex multicellular environments. Ultimately this work demonstrates the process and complexity involved in developing nanoparticulate systems meant to interact with incredibly complex intracellular environments.

Immunotherapy has been widely investigated in cancer, infectious diseases and allergies for prevention or amelioration of disease progression. In the case of vaccines, the key cellular target in stimulating an effective and appropriate immune response is the professional antigen presenting cell or dendritic cell (DC). Cancer vaccines are primarily aimed at the activation of a tumor-specific cytotoxic T lymphocyte (CTL) response whilst vaccines to allergies are aimed at reducing IgE responses. Such vaccines normally involve the administration tumor-associated antigens (TAAs) for cancer, or antigens (Ags) derived from infectious microbes and allergens in the case of allergies. Ags, whether derived from tumor or allergen, can be combined with adjuvants, that include immunostimulatory molecules recognized by the pathogen associated receptors expressed by DCs and can trigger the activation/maturation of DCs. Co-delivery of an appropriate adjuvant with an Ag can stimulate DCs to subsequently promote a robust Ag-specific CTL response which may favor anti-tumor immunity. Cancer vaccines have been widely investigated in the clinics as a complementary therapy to surgery, radiation and chemotherapy. Activation of CTLs against tumor cells that express TAAs could lead to the complete eradication of a cancer and prevent its reoccurrence. In this study I developed microparticles using a polyanhydride polymer prepared from 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) that has shown inherent adjuvant properties. I prepared 50:50 CPTEG:CPH microparticles encapsulating a model tumor Ag, ovalbumin (OVA), and synthetic oligonucleotide containing an unmethylated CpG motif, CpG, as an adjuvant. CpG has shown significant potential as an adjuvant for TAA-based vaccines leading to significant anti-tumor immune activity. I have shown that mice vaccinated with OVA-encapsulated 50:50 CPTEG:CPH microparticles developed OVA-specific CTL responses. These mice showed enhanced survival compared to the control treatment groups when challenged with OVA expressing tumor cells . In a more novel in-situ cancer vaccine, TAAs from dying tumor cells (caused by certain chemotherapeutic drugs) can be used as the source of Ags delivered to DCs. The presence of an adjuvant with dying cancer cells can assist in appropriate maturation of DCs so as to promote the generation of an effective tumor/TAA-specific CTL response against released TAAs. In this work I developed a therapeutic in situ tumor vaccine encapsulating a chemotherapeutic drug and CpG. Doxorubicin (Dox) is a widely used chemotherapeutic drug that induces tumor cells to undergo an immunogenic form of apoptosis. Sustained release of Dox in solid tumors of mice can cause the release of a variety of TAAs which can be presented by DCs and, in the presence of CpG, stimulate a strong anti-tumor CTL response. I prepared formulations of poly(lactic-co-glycolic acid) (PLGA) particles loaded with Dox and CpG which demonstrated sustained release of their cargo. I show that among various formulations of Dox and CpG, co-delivery of Dox and CpG in the same PLGA particles in-vivo showed the highest reduction in tumor growth and longest survival when compared to treatment groups of PLGA particles delivering Dox and CpG either alone or in combination. PLGA particles have also been investigated as a prophylactic vaccine delivery system that generates a robust Ag-specific CTL response. This system has been employed for the development of vaccines against various infectious diseases and allergies. However, there has been conflicting opinions regarding the optimum size of PLGA particles required to stimulate an active CTL response. Thus, I developed different sizes of PLGA particles encapsulating OVA and CpG to study the relationship of particle size with the magnitude of OVA-specific CTL responses. I showed that the degree of particle uptake and activation of DCs increased with decreasing size of PLGA particles. I also showed that immunization of mice with 300 nm sized particles demonstrated a higher proportion of OVA-specific CTLs and increased the secretion of IgG2a antibody responses. I also evaluated the efficacy of these particles with a clinically relevant Ag, Dermatophagoides pteronyssinus-2 (Der p2). Mice vaccinated with different sizes of PLGA particles loaded with CpG and coated with Der p2 displayed different magnitudes and types of immune activation against Der p2. The small sized particles decreased the airway hyperresponsiveness associated with allergy-induced asthma. The presence of CpG in the PLGA particle vaccines also reduced the airway hyperresponsiveness. This thesis research has contributed to the identification and development of a delivery system for Dox in combination with CpG which gives sustained release of these molecules within tumors and show the longest survival in tumor bearing mice. This study also optimized the size of PLGA particles for the delivery of vaccine to produce a robust Ag specific immune response for development of vaccination against intracellular diseases, cancer and allergy.

The emergence of antibiotic resistant bacteria has prompted the research into novel kinds of antibacterial small molecules and polymers. Nature has solved this issue with the use of cationic antimicrobial peptides, which act as nonspecific antibiotics against invading species. Herein, we have tried to mimic this general mechanism in a biocompatible and biodegradable polymer micelle based on the polymerization of naturally occurring amino acids lysine and phenylalanine linked to a PEG tether. This amphiphilic structure allows for the spontaneous collapse into stable nanoparticles in solution, which contains a hydrophilic outer layer and a hydrophobic core. Our polymers have shown activity against clinically relevant strains including Methicillin Resistant S. epidermidis, B. subtilis, K. pneumoniae, and P. aeruginosa. To further the application of our biopolymers, we have used them as drug delivery vehicles as well. First, we have used an anionic analogue based on glutamic acid to encapsulate a super hydrophobic drug Tanshinone IIA, and use it against a hepatoma bearing mouse model. Second, we have used a cationic analogue to form a complex with miRNA-139 and use it against a hepatoma bearing mouse model as well. In both cases, our PEG poly(amino acids)s have shown promising efficacy in drastically reducing the tumor size compared to the control only. Taken together, our results show that our nanoparticles have the potential to be versatile biomaterials as antibacterials as well as drug delivery vehicles in vivo.

Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated. Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies. Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity. Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.

Pulmonary and oral drug administrations are usually the preferred methods of delivery of active pharmaceutical ingredients.Generally,pulmonary drug formulations are more attractive compared to oral formulations since they consist of micron-sized powders with high surface area thus having faster onset of action,as well as minimizing the drug dosage and side effects.Oral insulin formulations,if achievable,would provide an alternative to injectable insulin,as the common drawbacks of injectable insulin are the multiple daily injections and the possibility of skin infections at the injection site. In this study,the feasibility of using dense gas particle processing techniques known as the Aerosol Solvent Extraction System (ASES),Gas Anti-Solvent (GAS)and High-Pressure Media Milling (HPMM)for pharmaceutical processing was assessed.The ASEStechnique,utilizing dense ethane,was employed to prepare insulin-lactose formulations for pulmonary administration whilst the GAS and ASES techniques,utilizing dense CO2,were employed to prepare microencapsulated formulations containing insulin and Eudragit?? S100 for oral administration.Furthermore,the HPMM technique,utilizing dense hydrofluocarbon (HFC)134a/227ea,was employed to prepare suspension Metered Dose Inhaler (MDI)formulations containing budesonide and various surfactants. The Fine Particle Fraction (FPF)of processed insulin without the presence of lactose was found to be 44%.In other words,44% of processed insulin delivered to the impactor stages (excluding the throat and neck)has aerodynamic diameter of less than 5??m.With the addition of lactose as carrier,the FPFof the insulin-lactose (1:1w/w)formulation increased to 64%.The increase in FPFwas attributed to the lower density of lactose particles compared to that of insulin particles to produce an intimate mixture with enhanced powder flowability and aerodynamic performance. Proteins for oral delivery should ideally be formulated with acid-resistant polymer as a protective coating to protect against enzymatic degradation in the stomach.Eudragit?? S100,which is insoluble or almost impermeable at pH 1-4and soluble at pH 5-7,was used to prepare oral insulin formulations.The insulin release at pH 3was sustained by the Eudragit?? S100coating and the encapsulation efficiency of insulin??Eudragit?? S100formulations varied between 6% and 24% depending on the initial drug to polymer ratio. One of the major therapies utilizing metered dose inhaler formulations in the treatment of asthma has been studied using the HPMM process.The HPMM process has been demonstrated to be an efficient milling process for the enhancement of the physical stability and aerodynamic performance of budesonide in HFC-134a/227ea propellant formulations.No significant change in physical stability was observed in the formulations for 2 weeks.

Since its discovery 15 years ago there has been an explosion of research in the field of DNA immunisation. Unfortunately despite early promises that DNA immunisation had the potential to cure almost any infectious disease, autoimmune disease or even cancer, progress towards clinical trials has been slow. This has been due in part to the huge range of permutations possible in delivering the DNA. One approach is to deliver the DNA by gene gun. Gene gun delivery is a very efficient way of transfecting cells however also has a number of possible disadvantages. These drawbacks include a weak immunogenicity in larger animals as well as the tendency to bias towards the development of a strong type 2 response. In an effort to enhance antigen-specific immune responses and counter the type 2 polarisation of gene gun delivery, a series of DNA vaccines were created where the extracellular portion of the hemagglutinin (HA) gene from influenza A/PR8/34 virus was genetically fused the type 1 cytokines IFNγ, IL-12 and IL-23. Interleukin-23 has been recently discovered and even though both IL-12 and IL-23 contain the p40 subunit they seem to have dissimilar functions. The vaccine constructs were first tested in cellular assays in vitro to ensure correct production and biological activity of the attached cytokines. They were then delivered in various combinations to groups of BALB/c mice to test development of immune responses and the effect of different delivery regimes. Finally mice were immunised then challenged with live influenza virus to determine the different DNA vaccines� protective efficacy. DNA vaccines containing the HA gene alone (pHA) or fused to IFNγ (pIFNγHA), IL-12 (pIL-12HA) or IL-23 (pIL-23HA) were successfully constructed. The fusion of the HA gene to the genes for IFNγ, IL-12 or IL-23 did not significantly disturb the structure of the antigen or prevent the biological actions of the cytokines. Mice immunised three times with pHA had high titres of serum IgG1 antibody and their splenocytes produced approximately equal amounts of IFNγ and IL-5. Co-delivery of IFNγ was unable to alter immune responses regardless of whether it was delivered at the first, last or during all immunisations. Surprisingly co-delivery of IL-12 acted to suppress both antibody and cellular immune responses, possibly through an IFNγ/nitric oxide feedback loop. On the other hand co-delivery of IL-23 tended to enhanced immune responses and, while it did not significantly alter the type 1 to type 2 balance, it was able to increase the ability of mice to clear live influenza virus from their lungs when they were challenged 26 weeks after immunisation. This protection was associated with increased levels of neutralising antibody in the serum of pIL-23HA immunised mice. This research has illuminated several of the pitfalls in the development of DNA vaccines and the use of cytokine as adjuvants. However it has also broadened our understanding of IL-23 and implies that IL-23 could be effectively used to increase the development of longterm immunity after immunisation.

Purpose: This thesis aimed to gain a better understanding of the effects of salts in modifying in vitro phase behaviour of lecithin and cholesterol solid implants and to obtain further information on in vitro protein release and stability. Methods: Raman spectroscopy and partial least squares regression (PLSR) were used to investigate lecithin-cholesterol molecular interactions as a function of method of preparation. Lipid-salt interactions were studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and Raman spectroscopy using principal component analysis (PCA). In vitro release of bovine serum albumin (BSA), a model protein, from lecithin and lecithin:cholesterol implants comprising 10 and 30% NaCl and CaCl₂ were performed. Size exclusion (SE) HPLC was used for quantitative and qualitative analysis of the released BSA. On hydration, changes in phase behaviour and implant morphology were studied by ATR spectroscopy and light microscopy. SE-HPLC, ATR and fluorescence spectroscopy were used to evaluate the structure of unreleased BSA. Protein adsorption on lipid films was studied by flow through ATR spectroscopy. Increased amide II peak area upon recirculation of BSA in salt solutions over hydrated lecithin and lecithin:cholesterol films cast on ZnSe prisms was used to quantify the deposition of BSA onto the lipid surfaces. Results: Shifts in the Raman spectra suggested the lecithin headgroup may be involved in lecithin-cholesterol interactions. Greater R� and root mean square error of cross validation in the calibration curves of physical mixing and heating (120�C) methods reflected poor mixing in these preparations. The mean absolute residue and mean Mahalanobis distance values from the physical mixing and granulation methods indicated their spectral similarity and comparable level of lecithin-cholesterol interactions. Calcium exhibited stronger affinity for phospholipids than sodium and it induced headgroup hydration and reorganisation upon binding. PCA of ATR spectra was sensitive to cholesterol addition, calcium binding and method of preparation whilst PCA of Raman spectra only differentiated the presence of cholesterol. In vitro release of BSA from implants produced from wet granulation mixtures of lecithin and lecithin:cholesterol in the absence of salt showed retention of a high monomer content and the release profiles were similar to the literature. Cholesterol increased the swelling, induced phase transformation of lecithin and, subsequently, reduced the BSA release. Salts only slightly modified the BSA release from the lecithin implants. In contrast, for lecithin:cholesterol matrices salts greatly enhanced implant swelling, induced the formation of hydrated lecithin of heterogeneous size and inhibited the in vitro BSA release. Analyses of the protein showed increased aggregation of BSA with a high retention of native structure while retained within the swollen matrices. ATR spectra suggested that salts promoted protein adsorption onto hydrated lecithin surfaces and the effects depend on salt types (NaCl > CaCl₂) and concentration (0.1 M > 1.0 M) but not on lecithin:cholesterol surfaces. Conclusion: PLSR and PCA can be used to investigate molecular interactions in the solid lipid matrices. In lecithin:cholesterol implants, salts modified the phase behaviour of lecithin which resulted in enhanced swelling, formation of hydrated lecithin of altered morphology and inhibition of in vitro BSA release.

Purpose: The aim of this thesis was to investigate a suitable formulation for the topical delivery of growth factors to chronic wounds, and then to determine the concentrations reached within an animal wound model. A secondary aim was to determine if the chosen growth factor was present at levels able to stimulate the production of other cytokines, specifically IL-1β and MCP-1. Methods: An in vitro testing apparatus was designed and made and the release of model actives [bromophenol blue (BPB) and horseradish peroxidase (HRP)] from gels and films of hydroxylpropylmethylcellulose (HPMC) (E4M CR, K4M CR and E10M CR) was determined. In this study, Fibroblast Growth Factor -2 (FGF-2) (0.3 [mu]g) was incorporated into three formulations (solution, gel and dried gel film on Melolin[TM] backing) and together with a control formulation were administered to punch biopsy wounds in rats. The in vivo release was followed over three time periods (two, five and eight hours) and the amount of FGF-2 at various wound depths was quantified by ELISA. Two biological markers IL-1β and MCP-1 were quantified using ELISA. The FGF-2 was additionally tagged with a fluorescent dye so that visualisation of the penetration could be obtained via confocal microsopy. Results: For the HPMC gels, the more viscous gel (E10M) provided a greater diffusional barrier and slowed the release of BPB (12 � 3.5 [mu]g/min compared with 16 � 1.7 [mu]g/min and 18 � 1.4 [mu]g/min for K4M and E4M respectively). However, when HPMC was formulated as a dried film a burst release was seen and release of BPB was slowest from the more rapidly hydrating K4M. With the larger model active HRP, there was a slower diffusion through the gel barrier formed upon film hydration, such that time of 100% release was up to 300 minutes compared to approximately 60 minutes for BPB. When the film was dried onto a supportive backing, the initial burst release was minimised as the film did not break apart on contact with the wound, and hence film integrity was maintained and release prolonged. The in vivo studies showed that, two hours after application, the highest concentration of FGF-2 was seen in the surface granulation tissue of rats that received the solution formulation (2280 � 790 pg/g). The concentration decreased with increasing tissue depth but was significantly greater than the saline control in the surface granulation and subcutaneous fat layers (p<0.05). Tissue concentrations following application of the gel and film formulations were only marginally greater than control in the surface granulation layer. After eight hours, rats that received the solution retained elevated surface tissue concentrations (surface granulation and subcutaneous fat) of FGF-2. Repeated measures ANOVA using a general linear model showed statistically significant differences in the mean FGF-2 level with respect to formulation and length of time of application of the formulation (p<0.05). In terms of other cytokines, there was a release of both IL-1β and MCP-1 in all groups, immediately post-wounding, probably in response to cellular damage. After eight hours, the film formulation appeared to elevate IL-1β levels which may be useful to drive wound healing. Confocal microscopy images showed diffuse distribution of FGF-2 eight hours after application of the solution formulation after eight hours and that with the gel formulation FGF-2 initially aggregated at the wound surface. Conclusion: In vitro experiments investigating the effect of hydration rate and viscosity of HPLC polymers allowed a formulation to be chosen for further in vivo study. Elevated FGF-2 could be measured in superficial wound tissues up to eight hours after application of a solution. However, application of a comparable amount of FGF-2 in HPMC gels or films did not produce appreciable elevations in FGF-2 in wound tissues, although confocal microscopy indicated the penetration of FGF-2 into the wound for up to eight hours.