In vitro enzymatic stability of dendritic peptides.

PEGylated polyamidoamine (PAMAM) dendrimers as drug carriers have been a topic of interest because of their biomedically favorable features, including minimal toxicity, reduced immunogenicity, and excellent solubility in aqueous and most organic solutions. A PEG shell on dendrimer surface may provide steric hindrance, known as stealth properties of PEG, to stabilize drug molecules to be delivered. In this article, the effects of PEG and coupling sequence of drug, PEG, and dendrimer in modulating the stability of delivered drug molecules were evaluated. N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was chosen as a model peptide. Dendritic peptides, that is, peptide-dendrimer, peptide-PAMAM-PEG, and peptide-PEG-dendrimer, were constructed based on Starbursttrade mark G3.0 PAMAM dendrimer and characterized by (1)H-NMR spectroscopy. Hydrolysis of dendritic peptides was catalyzed by alpha-chymotrypsin in pH 7.4 PBS buffer containing 5% DMF (v/v) at room temperature. The enzymatic stability of dendritic peptides was peptide-PAMAM-PEG > peptide-PAMAM > free peptide > peptide-PEG-PAMAM. The ratio of PEG/peptide could be reduced for increasing peptide loading while maintaining the delivered peptides' relatively high enzymatic stability. The quantitative analysis of dendritic peptide/enzyme interactions provided the understandings of the molecular structure/stability relationships of dendrimer/drug for the design of an optimal PEGylated dendrimer-based drug-delivery system.

Extended release of a novel antidepressant, venlafaxine, based on anionic polyamidoamine dendrimers and poly(ethylene glycol)-containing semi-interpenetrating networks.

The multiple daily administration of venlafaxine, a novel third-generation antidepressant, was reduced based on polyamidoamine and polyethylene glycol (PEG)-containing semi-interpenetrating network (IPN), respectively. Venlafaxine was covalently linked to water-soluble G2.5 anionic polyamidoamine dendrimer via a hydrolyzable ester bond. Semi-IPN hydrogels were prepared by crosslinking acrylamide in the presence of PEG, and venlafaxine with predetermined amounts was loaded in situ. Dendrimer-venlafaxine conjugate and semi-IPNs were characterized by proton nuclear magnetic resonance and Fourier transform infrared, respectively. The effect of PEG concentration and molecular weight was studied and discussed for an optimal controlled release.

Improving delivery of hydrophobic drugs from hydrogels through cyclodextrins.

A simple and effective technique of improving delivery of hydrophobic drugs from swellable systems is presented. Conventional methods of drug loading in hydrogel systems are limited by the characteristics of the pharmacological agent. The approach we present uses complexants to modulate drug release. Crosslinked poly(ethylene glycol) (PEG) hydrogels were synthesized, characterized, and used for vascular applications. The release of cyclosporine (CyA) from PEG hydrogels is significantly altered by the sterilization techniques. It was hypothesized that the release of CyA from PEG hydrogels can be modulated by using complexants. A cyclodextrin-CyA complex solution was prepared and used for drug loading. The sterilized PEG hydrogels that were loaded using the cyclodextrin-CyA complex solution had favorable release characteristics compared with the release from PEG hydrogels that were loaded using the conventional technique. Hence, drug release from swellable systems can be tailored by the application of this strategy.

Modeling of drug release from polymeric delivery systems--a review.

Polymeric drug delivery platforms have been receiving increasing attention in the past decade. The pharmaceutical industry is evaluating modes of delivery for their prized therapeutics at every step of the design cycle. Not only can the drug delivery platform transport drug molecules effectively, it can also improve patient compliance, offer greater patient convenience, and extend product lifecycles as patents expire. A large number of successful drug delivery systems have been developed as a result of an almost arbitrary selection of constituents and configurations. However, the development of advanced drug delivery systems relies on a judicious and careful selection of components, configurations, and geometries, which can be facilitated through mathematical modeling of controlled release systems. Mathematical modeling aids in predicting the drug release rates and diffusion behavior from these systems by the solution of an appropriate model, thereby reducing the number of experiments needed. It also aids in understanding the physics of a particular drug transport phenomenon, thus facilitating the development of new pharmaceutical products. The objective of this article is to review the spectrum of mathematical models that have been developed to describe drug release from polymeric controlled release systems. The mathematical models presented in this article have been grouped under diffusion controlled systems, swelling controlled systems, and erosion controlled systems as proposed by Langer and Peppas. Simple empirical or semi-empirical models and complex mechanistic models that consider diffusion, swelling, and erosion processes simultaneously are presented.

Polyphosphates and other phosphorus-containing polymers for drug delivery applications.

Poly(phosphate ester)s, polyphosphonates, and polyphosphazenes are three classes of phosphorus-containing polymers that have received wide attention over the past decade for their utility in biomedicine and tissue engineering. These three families of polymers can lead to a number of subclasses of polymers with varied properties. Significant research in this area has led to niche polymers with morphologies ranging from viscous gels to amorphous microparticles for utility in drug delivery. Furthermore, the pentavalency of phosphorus offers the potential for covalent linking of the drug. The classes of polymers discussed in this review are being explored in human clinical trials for vaccine delivery as well as delivery of oncolytic and CNS therapeutics. More applications in the areas of DNA delivery and tissue engineering are also being explored.

Polymeric sustained local drug delivery system for the prevention of vascular intimal hyperplasia.

Anastomotic intimal hyperplasia (IH) is a major cause of both autologous vein and synthetic vascular graft failure. We have previously published data suggesting that cyclosporin may reduce the development of IH in a canine model. However, systemic administration of cyclosporin could create serious adverse effects. Therefore, it is our long-term goal to test the hypothesis that the controlled local release of cyclosporin from a polymeric vascular wrap will prevent the development of IH. To test this hypothesis, we developed a controlled release vascular wrap (sheet/ring) using a poly(ethylene glycol) (PEG) hydrogel. Sterilization of the polymers was performed using the ethylene oxide and hydrogen peroxide sterilization methods. It was found that except for one combination (8000 molecular weight and 1:1 crosslinking ratio), the differences in the swelling ratios for the sterilized and unsterilized hydrogels were not statistically significant. Release studies from unsterilized and ethylene oxide-sterilized PEG hydrogels were conducted. It was found that release lasted for approximately 50 h for sterilized as well as unsterilized PEG hydrogels. Acute animal studies, to test the deployment of both the polymeric sheets and rings to the adventitial surface of native arteries and veins, were completed successfully.

Polyethylene glycol-polyamidoamine dendritic micelle as solubility enhancer and the effect of the length of polyethylene glycol arms on the solubility of pyrene in water.

Unimolecular dendritic micelles designed as solubility enhancers were obtained by coupling polyethylene glycol (PEG) to Starburst polyamidoamine (PAMAM) dendrimers. Micelles-750, -2000, and -5000 have a generation 3.0 dendrimer core (32 primary amine end groups) and PEG arms with molecular weights of 750, 2000, and 5000, respectively. The conjugate of dendrimer core and PEG was characterized by MALDI-TOF MS and 1H NMR. 1H NMR was also used to estimate the average number of PEG arms on each dendrimer molecule. A typical hydrophobic compound, pyrene, was sonicated in an excess amount together with micelles at 50 degrees C for 6 h to produce its saturated water solution. The change of the solubility of pyrene was monitored at 334 nm, its maximum adsorption wavelength, by UV-VIS spectra. Concentrated micelles tended to dissolve more pyrene. However, there is no obvious linear relationship between micelle type and the amount of pyrene entrapped within micelles. Micelle-2000 could solubilize more pyrene than micelle-750. It is hypothesized that micelle-5000 did not solubilize more pyrene than micelle-2000 because of the PEG shell disruption by adjacent interpenetration of individual micelles when PEG arm length increased.

Sustained local drug delivery from a novel polymeric ring to inhibit intimal hyperplasia.

The long-term clinical success of autologous vein and synthetic vascular grafts are limited because of the development of anastomotic intimal hyperplasia (IH). We have previously published data suggesting that cyclosporine (CyA) may reduce the development of IH in a canine model (Hirko et al., J Vasc Surg 1993;17:877-887). However, systemic administration of CyA could create serious adverse effects. Therefore, it is our long-term goal to test the hypothesis that the controlled local release of CyA from a polymeric vascular wrap would prevent the development of IH. To test this hypothesis, we developed a controlled release polymeric ring that could be placed around anastomotic sites to deliver therapeutic drugs locally. The ring is a composite polymeric device consisting of poly(DL-lactide-co-glycolide) (PLGA) microspheres embedded in a poly(ethylene glycol) hydrogel. Several in vitro studies were conducted to evaluate the effects of different sterilization procedures on the properties of the device. It was determined that gamma sterilization was the preferred sterilization method of choice for this device. In vivo studies were conducted on a swine model to evaluate the biocompatibility of the ring. The histological findings of the ring implants at 2 and 4 weeks demonstrate the biocompatibility of this device.

Synthesis and characterization of L-tyrosine based polyurethanes for biomaterial applications.

The use of amino acid based polymers for biomaterial applications enhance biocompatibility and ensure biodegradability. Two polyurethanes based on L-tyrosine based diphenolic dipeptide, desaminotyrosyl tyrosine hexyl ester as chain extender are synthesized with polyethylene glycol (PEG) and polycaprolactone diol (PCL) as soft segment and hexamethylene diisocyanate as diisocyanate. The chemical structure and molecular characteristics of the polymers were studied by 1H NMR, FTIR, and gel permeation chromatography. Results of DSC and TGA analysis were used for examining the thermal behavior of the polyurethanes. In addition, DSC results were used to analyze the morphology of the polymers, which shows characteristic microphase behavior of the polyurethanes. The tensile properties of the polyurethanes are primarily controlled by the soft segment and are higher in PCL based polymers. Contact angle, water vapor permeation, release of model drug, and water absorption characteristics of the polymers were studied and analyzed in terms of structure of the polyurethanes. In vitro degradation studies show that PEG based polyurethane is more degradable than PCL based polyurethane. The difference in the soft segment structure offers significant variation in the properties of the polyurethanes. These polyurethanes show the potential for use in a variety of biomaterial applications including tissue engineering.

Nanospheres formulated from L-tyrosine polyphosphate as a potential intracellular delivery device.

Current delivery devices for drugs and genes such as films and microspheres are usually formulated from polymers that degrade over a period of months. In general, these delivery systems are designed to achieve an extracellular release of their encapsulated drugs. For drugs that require interaction with cellular machinery, the efficacies of both macroscopic and microscopic delivery systems are normally low. In contrast, nano-sized drug delivery vehicles could achieve high delivery efficiencies, but they must degrade quickly, and the delivery system itself should be nontoxic to cells. In this aspect, biodegradable nanospheres formulated from l-tyrosine polyphosphate (LTP) have been produced from an emulsion of oil and water for the potential use as an intracellular delivery device. Scanning electron microscopy (SEM) and dynamic laser light scattering (DLS) show that LTP nanospheres possess a diameter range between 100 and 600 nm. SEM reveals nanospheres formulated from LTP are spherical and smooth. Additionally, DLS studies demonstrate that nanospheres degrade hydrolytically in 7 days. Confocal microscopy reveals LTP nanospheres are internalized within human fibroblasts. Finally, the cell viability after exposure to LTP nanospheres and determined with a LIVE/DEAD Cell Viability Assay is comparable to a buffer control. In conclusion, our nanospheres have been shown to be nontoxic to human cells, possess the appropriate size for endocytosis by human cells, and degrade within 7 days. Therefore LTP nanospheres can be used for a sustained intracellular delivery device.

Electron spin resonance studies of the effects of sterilization on poly(ethylene glycol) hydrogels.

The effects of several sterilization procedures on a poly(ethylene glycol) (PEG) hydrogel have been examined by electron spin resonance (ESR) spectroscopy. The crosslinked polyurethanes were synthesized by reacting PEG with a tri-functional isocyanate. The free radical concentration of unsterilized, ethylene oxide (EtO), hydrogen peroxide (H(2)O(2)), and gamma sterilized hydrogels were monitored over time. Free radical presence was observed for all the treatments, unsterilized and sterilized PEG hydrogels. The unsterilized and the EtO sterilized samples elicited similar levels of free radical intensity whereas, the H(2)O(2) and gamma sterilized samples had a significantly higher free radical concentration. The spectra reveal overlapping resonances of a peroxy and a triphenylmethyl radical. The concentration of the free radicals increase for all the treatments over time except for the gamma sterilized sample. The increase is significantly higher in the H(2)O(2) sterilized sample. A tentative model is proposed to explain the reaction pathway leading to the production of the free radicals. The observed increases in the free radical concentrations of the EtO and hydrogen peroxide sterilized hydrogels over a five-month-period make it difficult to predict properties that are affected by free radical concentrations. In that light, gamma sterilization, that does not induce a change in free radical concentrations over a five month period, could be the sterilization method of choice for PEG hydrogels that could potentially be stored for undetermined periods of time prior to application.

Stealth dendrimers for drug delivery: correlation between PEGylation, cytocompatibility, and drug payload.

It is advantageous to utilize low generation polyamidoamine (PAMAM) dendrimers for drug delivery because low generations (generation 4.0 or below) have more biologically favorable properties as compared to high generations. Nevertheless, modification of low generation dendrimers with PEG to create stealth dendrimers is still necessary to avoid potential side effects by long term accumulation. However, low generation dendrimers have much fewer surface sites for drug loading as compared to higher generations. To efficiently utilize low generation dendrimer-based stealth dendrimers for drug loading, PEGylation needs to be optimized. In this study, we synthesized a series of stealth dendrimers based on low generation Starburst PAMAM dendrimers (i.e., G2.5, G3.0, G3.5, and G4.0) and quantitatively assessed PEGylation efficacy in modulating cytocompatibility of low generation PAMAM dendrimers. Cytocompatibility of stealth dendrimers was examined using endothelial cells. The results showed that PEGylation degree on low generation dendrimers could be dramatically reduced to leave as many unoccupied surface groups as possible for drug loading, while maintaining the drug carrier cytocompatibility. 3PEGs-G3.0 and 10PEGs-G4.0 were considered initially optimized stealth dendrimers that would be further modified to deliver drugs of interest. Correlation of PEGylation, cytocompatibility, and drug payload allowed us to optimize low generation dendrimer-based stealth dendrimers for drug delivery and advance the understanding of structure-property relationship of stealth dendrimers.

Effects of sterilization on poly(ethylene glycol) hydrogels.

The past few decades have witnessed a dramatic increase in the development of polymeric biomaterials. These biomaterials have to undergo a sterilization procedure before implantation. However, many sterilization procedures have been shown to profoundly affect polymer properties. Poly(ethylene glycol) hydrogels have gained increasing importance in the controlled delivery of therapeutics and in tissue engineering. We evaluated the effect of ethylene oxide (EtO), hydrogen peroxide (H(2)O(2)), and gamma sterilization of poly(ethylene glycol) hydrogels on properties relevant to controlled drug delivery and tissue engineering. We observed that the release of cyclosporine (CyA) (an immunosuppressive drug that is effective in combating tissue rejection following organ transplantation) was significantly affected by the type of sterilization. However, that was not the case with rhodamine B, a dye. Hence, the drug release characteristics were observed to be dependent not only on the sterilization procedure but also on the type of agent that needs to be delivered. In addition, differences in the swelling ratios for the sterilized and unsterilized hydrogels were statistically significant for 1:1 crosslinked hydrogels derived from the 8000 MW polymer. Significant differences were also observed for gamma sterilization for 1:1 crosslinked hydrogels derived from the 3350 MW polymer and also the 2:1 crosslinked hydrogels derived from the 8000 MW polymer. Atomic force microscopy (AFM) studies revealed that the roughness parameter for the unsterilized and EtO-sterilized PEG hydrogels remained similar. However, a statistically significant reduction of the roughness parameter was observed for the H(2)O(2) and gamma-sterilized samples. Electron spin resonance (ESR) studies on the unsterilized and the sterilized samples revealed the presence of the peroxy and the triphenyl methyl carbon radical in the samples. The gamma and the H(2)O(2)-sterilized samples were observed to have a much higher concentration of the radical pecies when compared with the EtO and the unsterilized samples.

Stealth dendrimers for antiarrhythmic quinidine delivery.

Dendrimers have been attracting growing attention because of their unique well-defined globular nanoscale architecture and numerous functional groups on the surface. Attachment of PEG to the dendrimer generates stealth dendrimers, which have promising structural advantages for drug delivery. In this study, synthetic methods were explored to deliver antiarrhythmic quinidine by stealth dendrimers. In particular, quinidine was covalently attached to anionic G2.5 and cationic G3.0 polyamidoamine (PAMAM) dendrimers via a glycine spacer, respectively. The resulting quinidine-PAMAM-PEG conjugates were characterized and confirmed by FT-IR and (1)H-NMR. In vitro hydrolysis was carried out in pH 7.4 PBS buffer at 37 degrees C to confirm the bioavailability of the conjugated quinidine.

L-tyrosine-based backbone-modified poly(amino acids).

Tyrosine-based pseudo-peptide polymers, first introduced in 1987 by Kohn and Langer, have been identified for potential biomaterial applications. These materials combine the desired polypeptide properties of biocompatibility, biodegradability, non-toxicity, and non-immunogenicity with good processing properties including solubility, thermal stability, and moldability which arise from alternating non-amide bonds along the polymer backbone. This paper focuses on the analysis of two such polymers based on the natural amino acid L-tyrosine. Starting from L-tyrosine and its deaminated analogue, 3-(4-para-hydroxy)-phenylpropionic acid, a diphenolic structure containing an amide linkage, was synthesized following standard procedures of peptide synthesis. This diphenolic structure was then used as a monomer to synthesize a polyiminocarbonate using a cyanogen bromide-initiated reaction and a polycarbonate using a triphosgene-initiated reaction. The polyiminocarbonate has iminocarbonate linkages and the polycarbonate has carbonate linkages alternating with amide linkages in the respective polymer backbone. Analytical studies were performed regarding the feasibility of the reaction procedures, the physical properties of the polymers, and their degradation processes, to gain insight into the potential biomaterial applications of these polymers. These results independently reaffirm the studies published by Kohn et al. working on similar polymeric systems.

Penicillin V-conjugated PEG-PAMAM star polymers.

Starburst PAMAM dendrimers are potential carriers for drug delivery due to their unique structure. Drug-delivery scaffolds were designed and built up based on the polyethylene glycolpolyamidoamine (PEG-PAMAM) star polymer. Penicillin V was used as a model carboxylic group containing drug to conjugate with full- and half-generation PAMAM dendrimers. G2.5 PAMAM (with 32 carboxylic groups on the surface) dendrimers and G3.0 (with 32 primary amine groups on the surface) were typically chosen. There are two strategies given in the paper where a drug carrying a carboxylic group (e.g. penicillin V) was coupled to star polymer via amide and ester bonds, respectively. FT-IR, UV-Vis and 1H-NMR were used to characterize the intermediates and drug-star polymer conjugates. A single-strain bacterium, Staphylococcus aureus, was grown up for penicillin-conjugated PEG-PAMAM (G3.0) star polymer activity test. The result verified the bioavailability of modified penicillin after the ester bond was cleaved.