Model Protein Drug Research Articles

Preparation and characterization of spray-dried powders intended for pulmonary delivery of Insulin with regard to the selection of excipients

The aim of this study was to produce microparticles with optimal aerodynamic diameter for deep lung delivery (i.e., 1-3μm) of a protein drug intended for systemic absorption, using a combination of generally regarded as safe (GRAS) excipients. Based on the preliminary experiments, mannitol, l-alanine, sodium alginate, chitosan and dipalmitoylphosphatidilcholine (DPPC) were chosen as excipients and human insulin as a model protein drug. Dry powders were prepared by spray-drying. Powders with varying yields (29-80%) and low tapped densities (0.22-0.38 g/cm(3)) were obtained. Scanning electron microscopy (SEM) revealed distinctive particle morphologies among formulations from isolated spherical to highly folded particles. Aerodynamic properties were assessed by next generation impactor (NGI). Mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF) ranged from 2.1 to 4.6 μm and 46 to 81%, respectively. A comparative study of protein release from microparticles was conducted in vitro using an open membrane system with more than 50% cumulative release in all formulations which followed different kinetic models. Insulin's integrity was investigated by spectrofluorimetry and electrophoresis, and no tangible changes were observed in the structure of insulin. Of the formulations studied, the third, containing mannitol/sodium alginate/insulin/sodium citrate showed promising characteristics, optimal for systemic delivery of proteins via deep lung deposition.

In vitro evaluation of spray‐dried chitosan microspheres crosslinked with pyromellitic dianhydride for oral colon‐specific delivery of protein drugs

ABSTRACTChitosan microspheres have been prepared using a spray‐drying method, and crosslinked with pyromellitic dianhydride. The chemical structure of the modified chitosan was characterized by FTIR spectroscopy and solid state 13C NMR analysis. The particle size and morphology of the crosslinked chitosan were investigated. These microspheres were evaluated for colon‐specific delivery of bovine serum albumin (BSA) as a model protein drug. The results indicate that the drug was released as follows: 37.1 ± 2.8% after 2 h in SGF, 73.1 ± 4.8% after 8 h (2 h in SGF+ 6 h in SIF), and 80.9 ± 4.1% after 12 h in SCF. The effect of β‐glucosidase on the drug release was also examined. The encapsulation efficiency was decreased from 88.4 ± 3.1% to 62.8 ± 2.9%, with increasing BSA concentration. Loading capacity was significantly increased from 6.3 ± 0.3% to 41.8 ± 4.1% by increasing the initial BSA concentration. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40514.

Controlled Release of Protein from Biodegradable Multi-sensitive Injectable Poly(ether-urethane) Hydrogel

The synthesis and characterization of multi-sensitive polymers for use as injectable hydrogels for controlled protein/drug delivery is reported. A series of biodegradable multi-sensitive poly(ether-urethane)s were prepared through a simple one-pot condensation of poly(ethylene glycol), 2,2'-dithiodiethanol, N-methyldiethanolamine, and hexamethylene diisocyanate. The sol-gel phase transition behaviors of the obtained copolymers were investigated. Experimental results showed that the aqueous medium comprising the multi-segment copolymers underwent a sol-to-gel phase transition with increasing temperature and pH. At a certain concentration, the copolymer solution could immediately change to a gel under physiological conditions (37 °C and pH 7.4), indicating their suitability as in situ injectable hydrogels in vivo. Insulin was used as a model protein drug for evaluation of the injectable hydrogels as a site-specific drug delivery system. The controlled release of insulin from the hydrogel devices was demonstrated by degradation of the copolymer, which is modulated via the 2,2'-dithiodiethanol content in the poly(ether-urethane)s. These hydrogels having multi-responsive properties may prove to be promising candidates for injectable and controllable protein drug delivery devices.

Glucose-sensitive polyelectrolyte nanocapsules based on layer-by-layer technique for protein drug delivery

The glucose-responsive nanocapsules [CS-NAC/p(GAMA-r-AAPBA)] were readily fabricated with modified chitosan (CS-NAC) and random glycopolymer poly(D-gluconamidoethyl methacrylate-r-3-acrylamidophenylboronic acid) p(GAMA-r-AAPBA) as the alternant multilayered polyelectrolyte hybrid shell via layer-by-layer self-assembly after etching the amino functionalized SiO2 spheres by NH4F/HF. The spherical and hollow structure of nanocapsules was confirmed by TEM analysis and there was no clear collapse found after removal of the sacrificial cores. The reversible zeta potential changes of the nanocapsule materials evaluated the reversible glucose sensitivity. Besides, this system demonstrated a good capacity for encapsulation and loading insulin entrapped in nanocapsules as model protein drug. A good biocompatibility of the material was confirmed by the cell viability. In vitro release of insulin experiments revealed that no obvious release was found in acidic condition and the release could be normally conducted at physiological pH. These results implied that it was feasible for nanocapsules to be used in controlled release drug delivery system.

Osmotic pressure driven protein release from viscous liquid, hydrophobic polymers based on 5-ethylene ketal ε-caprolactone: Potential and mechanism

In this study, the potential of low molecular weight, viscous liquid polymers based on 5-ethylene ketal ε-caprolactone for localized delivery of proteins via an osmotic pressure release mechanism was investigated. Furthermore, the osmotic release mechanism from viscous liquid polymers was elucidated. 5-Ethylene ketal ε-caprolactone was homopolymerized or copolymerized with d,l-lactide (DLLA) by ring-opening polymerization. Polymer hydrophobicity was adjusted by choice of initiator; hydrophobic polymers were prepared by initiating with octan-1-ol, while more hydrophilic polymers were prepared by initiating with 350g/mol methoxy poly(ethylene glycol) (PEG). Particles consisting of bovine serum albumin (BSA) as a model protein drug were co-lyophilized with trehalose at 50:50 and 10:90 (w/w) ratios and were mixed into the polymers at 1% and/or 5% (w/w) particle loading. The release and mechanism of release of BSA from the polymers were assessed in vitro. BSA was released in a sustained manner, with a near zero-order release profile and with minimal burst effect for 5–80days depending on the polymer’s hydrophilicity; the release was faster from the PEG initiated polymers than from the octan-1-ol initiated polymers. Increasing the particle loading from 1% to 5% (w/w) resulted in a more noticeable burst effect, but did not significantly increase the mass fraction release rate. This release behavior was determined to proceed as follows. Release from the polymer was triggered by the water activity gradient between the surrounding aqueous medium and the saturated solution, which forms when water is absorbed from the surrounding medium to dissolve a given particle. The generated pressure initiates swelling around the particle/polymer interface and creates a superhydrated polymer region through which the solute is transported by convection, at a rate determined by the osmotic pressure generated.

Characterization of physiochemical and biological properties of spherical protein crystals for sustained release

This study presents an exploration on extending the action of therapeutic proteins by crystallization strategy without new molecular entities generation. Recombinant human interferon α-2b (rhIFN), a model protein drug in this case, was crystallized using a hanging drop vapor diffusion method. A novel chelating technique with metal ions was employed to promote crystals formation. The physico-chemical characterization of the protein crystals, including morphology, particle size, X-ray diffraction, circular dichroism and biological potency evaluations were performed. In addition, the in vitro release behavior of rhIFN from crystal lattice suggested an exciting possibility of protein crystals as a long-acting formulation. The work described here demonstrates the possibility of spherical crystals of biomacromolecules for controllable delivery application of therapeutic proteins.

Biodegradable thermogelling poly(ester urethane)s consisting of poly(1,4-butylene adipate), poly(ethylene glycol), and poly(propylene glycol)

New biodegradable and thermoresponsive amphiphilic multiblock poly(ester urethane) copolymers consisting of poly(1,4-butylene adipate) (PBA), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) were synthesized. The chemical structures and molecular characteristics of the copolymers were confirmed by gel permeation chromatography and proton nuclear magnetic resonance spectroscopy. The copolymers had relatively high molecular weights ranging from 40k to 46k with narrow molecular weight distributions. The amphiphilic block copolymers could form micelles in water with very low critical micellization concentration (CMC) values that are dependent on the PBA content and temperature. The copolymers with higher PBA contents showed lower CMC values because the PBA segments increased the hydrophobicity of the copolymers. The thermodynamic parameters of the micellization process were studied through CMC dependence on temperature, and it was concluded that the micellization process is a spontaneous and entropy-driven process. The aqueous solutions of the copolymers could form thermosensitive hydrogels with very low critical gelation concentrations (CGCs) ranging from 5 to 8 wt%. Higher PBA contents resulted in lower CGCs and higher strength of the hydrogels, again because of the hydrophobicity of the PBA segments. The hydrolytic degradation studies showed that these hydrogels could degrade rapidly within a period of 40 days, which resulted in the mass loss of the hydrogels and the molecular weight decrease of the copolymers. The copolymer hydrogels could sustain the release of the bovine serum albumin (BSA) model protein drug up to a few weeks, and the protein release rate could be controlled by the PBA contents in the copolymers. The cell viability assay showed that the poly(PEG/PPG/PBA urethane)s did not elicit cytotoxic response against the L929 cells with the copolymer concentration up to 0.63 mg mL−1. The PBA-based polyurethane copolymers may be candidate biomaterials for promising biomedical applications.

Dual stimuli responsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogels based on a β-cyclodextrin crosslinker: synthesis, properties, and controlled protein release

pH- and thermal-sensitive hydrogels were synthesized by copolymerization of acrylic acid and N-isopropylacrylamide with a modified β-CD crosslinker in aqueous solutions. The swelling behaviors of the hydrogels at different temperatures and pHs were examined. The hydrogels shrink in acidic conditions or at temperatures above their collapse temperature and swell in neutral or basic media or at lower temperatures. These processes can be reversed by changing the pH or temperature. Using bovine serum albumin as the model protein drug, in vitro drug release experiments were carried out at different pH values and temperatures. The pH- and temperature-dependent release behaviors indicate that these materials show much promise as stimuli-responsive drug delivery carriers.

Development and Evaluation of Lyophilized Thiolated-Chitosan Wafers for Buccal Delivery of Protein

Annealed and non-annealed freeze-dried wafers from synthesised thiolated-chitosan have been developed and evaluated. Wafers were obtained by freeze-drying aqueous gels of the thiolated polymer incorporating per polymer weight, 10 % each of glycerol as plasticizer, D-mannitol as cryoprotectant and 50% BSA as model protein drug. The formulation was freeze-dried with or without the process of annealing. Texture analyzer was employed to investigate the in vitro mucoadhesive properties in tensile mode, residual moisture content by thermo-gravimetric analysis (TGA) while hydration capacity and drug release studies were performed in 0.1M PBS. Microscopic architecture was examined using scanning electron microscopy (SEM). Differential scanning calorimetry (DSC) was employed for protein phase separation studies and conformational stability by attenuated total reflectance-Fourier transform infra-red spectroscopy (ATR-FTIR) and confirmed with Circular dichroism (CD). The annealing process led to wafers with increased ease of hydration, improved in vitro mucoadhesive characteristics and enhanced BSA release, without affecting the conformational stability of the protein due to the presence of a cryoprotectant. These results show the potential application of annealed freeze-dried thiolatedchitosan wafers for buccal mucosa delivery of protein based drugs.

Preparation of N, O-carboxymethyl chitosan-Human-Like Collagen Microspheres for Drug Delivery

In this investigation, the N, O-carboxymethyl chitosan (NOCC) was synthesized. A novel pH-sensitive hydrogel system composed of a water-soluble chitosan derivative (N,O-carboxymethyl chitosan, NOCC)and Human-like collagen (HLC) blended with phosphate was developed for controlling drug delivery and NOCC–HLC microspheres were prepared by the technology of electrostatic droplet generation technique. The release profiles of a model protein drug (bovine serum albumin, BSA) from test hydrogels were studied in simulated gastric and intestinal media.

Calcium Phosphate Nanocarriers Dual‐Loaded with Bovine Serum Albumin and Ibuprofen: Facile Synthesis, Sequential Drug Loading and Sustained Drug Release

A simple and green strategy is reported for the preparation, drug loading, and release properties of a drug delivery system consisting of calcium phosphate (CP) nanocarriers dual-loaded with bovine serum albumin (BSA) and hydrophobic drug ibuprofen (IBU). The sequential loading of BSA and IBU in calcium phosphate nanocarriers and in vitro simultaneous release of BSA and IBU are realized and investigated. In this method, BSA, which is used as a model protein drug, is encapsulated in situ in calcium phosphate nanocarriers. Subsequently, the typical hydrophobic drug IBU is loaded in the BSA/CP drug delivery system, forming the IBU/BSA/CP dual drug delivery system. The experiments reveal that the preloaded BSA not only reduces the cytotoxicity of calcium phosphate nanocarriers but also significantly improves the IBU drug loading capacity in calcium phosphate nanocarriers and greatly extends the duration of drug release. Thus, the as-prepared IBU/BSA/CP dual drug delivery system is promising for drug delivery applications.

Protein encapsulation in polymeric microneedles by photolithography

Recent interest in biocompatible polymeric microneedles for the delivery of biomolecules has propelled considerable interest in fabrication of microneedles. It is important that the fabrication process is feasible for drug encapsulation and compatible with the stability of the drug in question. Moreover, drug encapsulation may offer the advantage of higher drug loading compared with other technologies, such as drug coating. In this study, we encapsulated a model protein drug, namely, bovine serum albumin, in polymeric microneedles by photolithography. Drug distribution within the microneedle array was found to be uniform. The encapsulated protein retained its primary, secondary, and tertiary structural characteristics. In vitro release of the encapsulated protein showed that almost all of the drug was released into phosphate buffered saline within 6 hours. The in vitro permeation profile of encapsulated bovine serum albumin through rat skin was also tested and shown to resemble the in vitro release profile, with an initial release burst followed by a slow release phase. The cytotoxicity of the microneedles without bovine serum albumin was tested in three different cell lines. High cell viabilities were observed, demonstrating the innocuous nature of the microneedles. The microneedle array can potentially serve as a useful drug carrier for proteins, peptides, and vaccines.

Succinylated Pullulan Acetate Microspheres for Protein Delivery

【In order to develop new protein carrier replacing poly(DL-lactic acid-co-glycolic acid) (PLGA) microspheres, succinylated pullulan acetate (SPA) was investigated to fabricate a long term protein delivery carrier. SPA microspheres loaded with lysozyme (Lys) as a model protein drug were prepared by a water/oil/water (W/O/W) double emulsion method. An acidity test of SPA copolymers after hydrolysis was performed to estimate the change of protein stability during releasing proteins from the microspheres. There was no pH change of SPA copolymers, but pH of PLGA polymers after hydrolysis was significantly decreased to around pH 2, indicating that the long-term stability of proteins released from SPA microspheres can be guaranteed. Loading efficiency of proteins into SPA microspheres was three times higher than those into conventional PLGA microspheres, indication of inducing stronger charge interaction between proteins and succinyl groups in SPA microspheres. Although initial burst behaviors were monitored in Lys-loaded SPA microspheres due to relatively strong hydrophilic succinyl segments in SPA microspheres, initial burst issues would be circumvented if the ratio of charge density of succinyl moieties and hydrophobic acetate groups is harmonically controlled. Therefore, in this study, a new attempt of protein delivery system was made and functional SPA was successfully confirmed as a new protein carrier.】

Engineered peptides with enzymatically cleavable domains for controlling the release of model protein drug from “soft” nanoparticles

Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that has been shown to be present in high concentrations during most tissue remodeling events, including disease states like active tumor sites, thus making it an attractive molecule for use in effecting local delivery of therapeutic molecules. Moreover, the use of non-toxic and biodegradable nanoparticles for controlled drug delivery is highly sought after. To this end, bovine serum albumin (BSA) nanoparticles (NPs) were stabilized with coatings formed using domains of varying sensitivity to MMP-2, viz. K 6GPQG/IASQK 6 and K 6HPVG/LLARK 6, lysine residues being used to facilitate peptide immobilization to the BSA NPs via electrostatic interactions, and peptide domains that have a high (HPVG/LLAR) and low (GPQG/IASQ) MMP-2 cleavage rate. The MMP-2-induced cleavage rates of these two domains (the position of action being noted with a “/”) have differing kinetics that can be used to provide a novel mechanism for facilitating the controlled release of molecules where local concentrations of MMP-2 are high. It was found that both surface concentration and cleavage domain type influenced the release of the model drug (BSA) from these NPs. This stratagem may provide a novel pathway for developing multi-functional coatings for controlling the local delivery of therapeutics at sites where the presence of various enzymes exist as a function of tissue state.

Submicron bioactive glass tubes for bone tissue engineering

Herein we describe a method to fabricate submicron bioactive glass tubes using sol–gel and coaxial electrospinning techniques for applications in bone tissue engineering. Heavy mineral oil and gel solution were delivered by two independent syringe pumps during the coaxial electrospinning process. Subsequently, submicron bioactive glass tubes were obtained by removal of poly(vinyl pyrrolidone) and heavy mineral oil via calcination at 600 °C for 5 h. Tubular structure was confirmed by scanning electron microscopy and transmission electron microscopy imaging. We examined the bioactivity of submicron bioactive glass tubes and fibers and evaluated their biocompatibility, using electrospun poly(ε-caprolactone) fibers – a bioinactive material – for comparison. The bioactivity of the glass tubes was examined in a simulated body fluid and they demonstrated the formation of hydroxyapatite-like minerals on both the outer and inner surfaces. In contrast, mineralization only occurred on their surface for bioactive glass solid fibers. Energy-dispersive X-ray data suggested that the bioactive glass tubes had a faster induction of mineral formation than the solid fibers. We demonstrate that the proliferation rate of mouse preosteoblastic MC3T3-E1 cells on bioactive glass tubes was comparable to that on solid fibers. We also show that bioactive glass tubes can be loaded with a model protein drug, bovine serum albumin, and that these structures exhibit delayed release properties. The bioactivity of released lysozyme can be as high as 90.9%. Taken together, these data suggest that submicron bioactive glass tubes could hold great potential for use in bone tissue engineering as well as topical drug or gene delivery.

Photo‐cross‐linked biodegradable thermo‐ and pH‐responsive hydrogels for controlled drug release

AbstractA new strategy was developed to prepare thermo‐ and pH‐sensitive hydrogels by the crosslinking of poly(N‐isopropylacrylamide) with a biodegradable crosslinker derived from poly(L‐glutamic acid). Hydrogels were fabricated by exposing aqueous solutions of precursor containing photoinitiator to UV light irradiation. The swelling behaviors of hydrogels at different temperatures, pHs, and ionic strengths were examined. The hydrogels shrank under acidic condition or at temperature above their collapse temperature and would swell in neutral or basic media or at lower temperature. These processes were reversible as the pH or temperature changed. All hydrogels exhibited no weight loss in the simulated gastric fluid but degraded rapidly in the simulated intestinal condition. Bovine serum albumin were used as a model protein drug and loaded into the hydrogels. The in vitro drug release experiment was carried out at different pH values and temperatures. The pH and temperature dependent release behaviors indicated the promising application of these materials as stimuli‐responsive drug delivery vehicles. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Protein release parameters estimated with a flow system on zinc-containing apatite

Adsorption and desorption properties of proteins on zinc-containing apatite were successively monitored with a newly-developed flow system, and sustained-release ability of the apatite with different zinc contents was evaluated using protein release parameters we suggested. Three sustained-release parameters; initial desorption rate (rinit), time of desorption-completed (Tdes), and desorption constant (kd) were estimated with graphical analysis of dynamic desorption curves in a flow of 20 mM phosphate-buffered solution (PBS). Bovine serum albumin (BSA) of isoelectric point (pI) 4.8 and egg white lysozyme (LSZ) of pI 11.2 were employed as model protein drugs. Incorporation of zinc into hydroxyapatite changed desorption responses of the proteins. Zn(0.15), where the number in parentheses denoted the preparing molar ratio of Zn/Ca, showed the most sustained-release ability: less rinit, longer Tdes, and smaller kd. Furthermore, the adsorbed amounts of the proteins for Zn(0.15) were 1.5 ~ 4 times larger than Zn(0), which suggested that Zn(0.15) would be promising as a sustained-release carrier of protein drugs.

Novel protein-loaded chondroitin sulfate–chitosan nanoparticles: Preparation and characterization

In this study, the potential of chondroitin sulfate (ChS)–chitosan (CS) nanoparticles (NPs) for the delivery of proteins was investigated. ChS–CS NPs were prepared by ionic cross-linking of CS solution with ChS. The aggregation line, particle size and zeta potential were investigated as a function of the pH, weight ratio and concentration. The water content and formation yield of the NPs were measured by gravimetry. Results indicated that ChS–CS NPs showed a higher degree of ionic cross-linking and formation yield than sodium tripolyphosphate–CS NPs. Fluorescein isothiocyanate conjugate bovine serum albumin (FITC–BSA), a model protein drug, was incorporated into the ChS–CS NPs. The encapsulation efficiency was obviously increased with the increase in initial FITC–BSA concentration and was as high as 90%. In vitro release studies of ChS–CS NPs showed a small burst effect following a continued and controlled release. Cytotoxicity tests with Caco-2 cells showed no toxic effects of ChS–CS NPs. The ex vivo cellular uptake studies using Caco-2 and HEK-293 cells indicated that NPs were found to be endocytosed into the cells. In conclusion, ChS–CS NPs are a potential new delivery system for the transport of hydrophilic compounds such as proteins.

Encapsulation enhancement and stabilization of insulin in cationic liposomes

The purpose of this study was to enhance encapsulation efficiency and sustained-release delivery for parenteral administration of a protein drug. To reduce the administration frequency of protein drugs, it is necessary to develop sustained delivery systems. In this study, protein drug-loaded cationic liposomes were formulated with dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE/DOTAP/CH of 2/1.5/2. Five mol% of distearoylphosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) was added prior to encapsulation of the drug into liposomes. Insulin was chosen as a model protein drug and encapsulation efficiency was evaluated in various liposomes with and without DSPE-PEG. Scanning electron microscopy was used to examine the insulin-loaded cationic liposomes. Structural analysis was performed using spectropolarimetry. Additionally, the stability and cytotoxicity of insulin-loaded cationic liposomes were evaluated. Liposomes coated with DSPE-PEG showed higher insulin encapsulation efficiency than did those without DSPE-PEG, but not significantly. Moreover, among the liposomes coated with DSPE-PEG, those hydrated with 10% sucrose showed higher encapsulation efficiency than did liposomes hydrated in either phosphate-buffered saline or 5% dextrose. In vitro release of insulin was prolonged by cationic liposomes. Our findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of protein and peptide drugs to prolong efficacy and improve bioavailability.

The characterization of protein release from sericin film in the presence of an enzyme: Towards fibroblast growth factor-2 delivery

Aqueous preparations of silk protein (sericin) films were prepared to evaluate their biodegradation properties. In the absence of trypsin, sericin film swelled rapidly, kept its shape, and remained unaltered for 28 days or longer due to form β-sheet structures. In the presence of trypsin, sericin film gradually degraded; since the rate depended on the concentration of trypsin, the films likely underwent enzymatic hydrolysis. Sericin film incorporating the model protein drug fluorescein isothiocyanate–albumin (FA) also gradually degraded in the presence of trypsin and resulted in the sustained release of FA for 2 weeks or longer; in contrast, FA release was quite slow in the absence of trypsin. It is expected that sericin film has potential as a biodegradable and drug-releasing carrier. To evaluate the practical applicability of sericin film for the repair of defective tissues, fibroblast growth factor-2 (FGF-2) was incorporated into sericin films and the films were implanted on skull defects in rats. Whereas FGF-2 release was suppressed in the absence of trypsin in vitro, it appears that FGF-2, immobilized by ionic interactions between sericin and FGF-2, can be sustained-released in vivo from films incorporating 2500 or 250 ng of FGF-2 to support the growth of tissue around wounds.