Carriers For Protein Delivery Research Articles

Raspberry-like assembly of cross-linked nanogels for protein delivery

Raspberry-like assembly of nanogels (A-CHPNG) with a high potential as a carrier for protein delivery was prepared. Cross-linking of acrylate group-modified cholesterol-bearing pullulan nanogel (CHPANG) with thiol group-modified poly (ethylene glycol) (PEGSH) by Michael addition yielded A-CHPNG with narrow size distribution. The size of A-CHPNGs was controlled in the range of 40–120 nm by changing the concentration of CHPANG and PEGSH. A-CHPNG gradually degraded by hydrolysis under physiological condition and seemed to dissociate back to original nanogel. A-CHPNG encapsulated interleukin-12 (IL-12) efficiently (96%) and stably kept it in the presence of BSA (50 mg/ml). In addition, A-CHPNG had a high potential to maintain a high IL-12 level in plasma after subcutaneous injection in mice. Therefore, A-CHPNG is a promising carrier for long-term medications.

Assessment of poly(methacrylic acid-co-N-vinyl pyrrolidone) as a carrier for the oral delivery of therapeutic proteins using Caco-2 and HT29-MTX cell lines.

Hydrogels of poly(methacrylic acid-co-N-vinyl pyrrolidone) were synthesized and evaluated for their use as carriers for oral protein delivery. Insulin loading efficiencies were determined to be near 90% for carriers crosslinked with ethylene glycol dimethacrylate with corresponding weight incorporation levels near 12%. Although no insulin was released in gastric conditions, as desired, near instantaneous release occurred when the pH was raised to values typical of the intestinal area. Cytocompatibility studies with Caco-2 and Caco-2/HT29-MTX cultures demonstrated that microparticles did not elicit toxic effects at concentrations up to 5.0 mg/mL. Insulin transport studies revealed that the carriers did not disrupt the cell layer and thus did not change the insulin permeability in the apical-to-basolateral direction. Therefore, microparticles of this system were best suited for oral delivery of therapeutic agents that do not require transport facilitation.

Improving Oral Absorption of Samon Calcitonin by Trimyristin Lipid Nanoparticles

Solid lipid nanoparticles (SLN) composed of trimyristin (solid lipid) and poloxamer 407 (surfactant) were prepared by a w/o/w emulsion technique for the incorporation of Salmon calcitonin, and further explored as protein carriers for oral delivery. Trimyristin SLN showed a mean size diameter of 200 nm with an association efficiency for calcitonin of approx. 86%. The morphology of SLN was investigated by cryo-SEM and by AFM, revealing spheroid shape SLN with a smooth surface. The in vitro release of calcitonin occurred for a period of 8 h, under both gastric and intestinal simulated pH conditions, predicting suitable properties for oral administration. The pharmacological activity of the protein was evaluated following oral dosage of calcitonin-loaded SLN in rats. SLN lowered the basal blood calcium levels by up to 20% with 500 IU/kg dose sustaining hypocalcaemia over 8 h. The results indicate that incorporation of Salmon calcitonin into trimyristin SLN is a key factor for the improvement of the efficiency of such carriers for oral delivery of proteins.

Advancement in polymer therapeutics and characterization

The beauty of the discipline, polymers in drug delivery, is its longevity and self-transforming quality. Polymers have, for decades, performed a valuable function as excipients in tablet and capsule formulations, moving steadily into the parenteral arena as blood circulation time enhancers, and are now capable of offering advanced and sophisticated functions (such as drug targeting) to medicine. Polymers have unique cooperative properties that are not found with low-molecular weight compounds and therein lies the root of their success. Polymers are used as carriers for the delivery of drugs, proteins, targeting moieties, and imaging agents. Several polymers, polyethylene glycol, N-(2-hydroxypropyl) methacrylamide, and polylactide-co-glycolidecopolymers have been successfully utilized in clinical research. Recently, interest in polymer conjugation with biologically active components has increased remarkably as such conjugates are preferably accumulated in solid tumors and can reduce systemic toxicity. Further, it is essential to elucidate the structure–activity relationshipof a drug when it is conjugated with a polymer using different conjugation sites as this can vary the efficacy and mechanism of action when compared with its free form.This review will discuss the current advancement in drug targeting with polymers, smart polymers and recombinant polymers for drug delivery. Finally, it will also highlight on various methods of polymer characterization, including various techniques for polymer molecular weight measurement.

PEG-grafted chitosan nanoparticles as an injectable carrier for sustained protein release

The development of injectable nanoparticulate "stealth" carriers for protein delivery is a major challenge. The objective of this work was to investigate the possibility of achieving the controlled release of a model protein, insulin, from PEG-grafted chitosan (PEG-g-chitosan) nanoparticles (mean diameter 150-300 nm) prepared by the ion gelation method. Insulin was efficiently incorporated into the nanoparticles, and reached as high as 38%. In vitro release showed that it could control the insulin release by choosing the composition, loading and release temperature. We observed that the composition of the nanoparticle surface (C/O ratio) increased from 2.40 to 3.23, with an increase in the incubation time. Therefore, we concluded that during this time, insulin release from PEG-g-chitosan nanoparticles followed a diffusion mechanism in which erosion was negligible. The experiments also demonstrated that PEG-g-chitosan helped to maintain the natural structure of the protein entrapped in the nanoparticles.

Biodegradable Nanoparticles of Partially Methylated Fungal Poly(β‐L‐malic acid) as a Novel Protein Delivery Carrier

The preparation of nanoparticles from 75% methylated poly(beta-L-malic acid) is described. Their degradation in aqueous environments was examined and the influence of pH and lipase on the rate of hydrolysis was evaluated. Six proteins were used to estimate the loading efficiency of the nanoparticles. The amount of protein retained in the nanoparticles was found to depend on the acid/basic character of the protein. Protein release from the loaded nanoparticles upon incubation in water under physiological conditions encompassed polymer hydrolysis and happened steadily within 3-10 d. The activity loss of entrapped alpha-chymotrypsin caused by loading and releasing depended on the method used for loading.

Recent Advances and Patents on Solid Lipid Nanoparticles

Solid Lipid Nanoparticles (SLNs) have attracted increasing scientific and commercial attention as colloidal drug carriers during the last decade. They have emerged as a potential alternative compared to other colloidal systems like polymeric nanoparticles, liposomes and fat emulsions, as they have been claimed to combine their advantages but successfully overcome their drawbacks. SLN formulations are extensively developed and characterized for their in vitro and in vivo applications by various routes like parenteral, oral, pulmonary, ocular, and dermal. SLNs are being widely investigated as carriers for delivery of macromolecules like proteins, oligonucleotides and DNA. SLNs have already been taken up for medium and large scale production using two of its reported production methods. In fact, the first SLN based product has recently been introduced in the Poland market as a topically applied moisturizer. Newer methods for production of SLNs and their applications are being reported and patented. Nanostructured lipid carriers (NLC) are mixture of solid lipid and liquid lipid while Lipid Drug Conjugates (LDC) are water insoluble lipid carrier for loading of poorly lipid soluble drugs. These new generation of lipid nanoparticles have been claimed to overcome the shortcomings of SLNs. This article reviews the formulation, characterization, applications, and patents on the advances and research on SLNs, NLC and LDC.

Preparation and characterization of temperature-sensitive poly(N-isopropylacrylamide)-g-poly(L-lactide-co-ε-caprolactone) nanofibers

Biodegradable and elastic poly(L-lactide-co-e-caprolactone) (PLCL) was electrospun to prepare nanofibers, andN-isopropylacrylamide (NIPAAm) was then grafted onto their surfaces under aqueous conditions using60Co-γ irradiation. The graft yield increased with increasing irradiation dose from 5 to 10 kGy and the nanofibers showed a greater graft yield compared with the films. SEM confirmed that the PLCL nanofibers maintained an interconnected pore structure after grafting with NIPAAm. However, overdoses of irradiation led to the excessive formation of homopolymer gels on the surface of the PLCL nanofibers. The equilibrium swelling and deswelling ratio of the PNIPAAm-g-PLCL nanofibers (prepared with 10 kGy) was the highest among the samples, which was consistent with the graft yield results. The phase-separation characteristics of PNIPAAm in aqueous conditions conferred a unique temperature-responsive swelling behavior of PNIPAAm-g-PLCL nanofibers, showing the ability to absorb a large amount of water at < 32 °C, and abrupt collapse when the temperature was increased to 40 °C. In accordance with the temperature-dependent changes in swelling behavior, the release rate of indomethacin and FITC-BSA loaded in PNIPAAm-g-PLCL nanofibers by a diffusion-mediated process was regulated by the change in temperature. Both model drugs demonstrated greater release rate at 40 °C relative to that at 25 °C. This approach of the temperature-controlled release of drugs from PNIPAAm-g-PLCL nanofibers using gamma-ray irradiation may be used to design drugs and protein delivery carriers in various biomedical applications.

Development of amphiphilic γ-PGA-nanoparticle based tumor vaccine: Potential of the nanoparticulate cytosolic protein delivery carrier

Nanoscopic therapeutic systems that incorporate biomacromolecules, such as protein and peptides, are emerging as the next generation of nanomedicine aimed at improving the therapeutic efficacy of biomacromolecular drugs. In this study, we report that poly(γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent protein delivery carriers for tumor vaccines that delivered antigenic proteins to antigen-presenting cells and elicited potent immune responses. Importantly, γ-PGA NPs efficiently delivered entrapped antigenic proteins through cytosolic translocation from the endosomes, which is a key process of γ-PGA NP-mediated anti-tumor immune responses. Our findings suggest that the γ-PGA NP system is suitable for the intracellular delivery of protein-based drugs as well as tumor vaccines.

In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery

In this study, the effects of alginate modification on absorption properties of FITC-BSA loaded TMC nanoparticles were investigated on an in vitro model of GI epithelium (Caco-2 cells). The feasibility of applying TMC nanoparticles loaded with a model vaccine urease in oral vaccination was also studied. Alginate modified TMC nanoparticles showed higher FITC-BSA permeate efficiency than non-modified TMC nanoparticles. However, alginate modification barely had any effect on TMC nanoparticles’ property of decreasing TEER or enhancing drug paracellular transport. Mice s.c. immunized with urease loaded TMC nanoparticles showed highest systematic immune response (IgG levels) but the lowest mucosal response (secretory IgA levels). In the contrast, mice i.g. immunized with urease loaded TMC nanoparticles showed much higher antibody titers of both IgG and secretory IgA than those with urease solution or urease co-administrated with TMC solution. These results indicated that TMC nanoparticles are potential carriers for oral protein and vaccine delivery.

Surface crosslinking for delayed release of proxyphylline from PHEMA hydrogels

Delayed release systems find applications in chronotherapeutics and colon-specific delivery. They have also been considered suitable carriers for the oral delivery of peptides and proteins. In prior work, our research group has reported surface crosslinking as an effective technique to modify drug release profiles for poly(vinyl alcohol) (PVA) hydrogels, reducing the early burst effect in particular. Here, we demonstrate the feasibility of delayed release of proxyphylline from poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels via surface crosslinking. Studies on in vitro drug release and the morphology changes of PHEMA hydrogels during swelling and drug release showed that the highly surface crosslinked layers and the ruptures occurring in these layers during swelling were likely responsible for the delayed release. In addition, the initial burst was significantly reduced or even eliminated from the drug release profile for PHEMA to achieve near zero-order release by judicious selection of two surface crosslinking parameters: crosslinking reagent concentration and exposure time used for the surface crosslinking treatment.

Chitosan nanoparticle as protein delivery carrier—Systematic examination of fabrication conditions for efficient loading and release

Chitosan nanoparticles fabricated via different preparation protocols have been in recent years widely studied as carriers for therapeutic proteins and genes with varying degree of effectiveness and drawbacks. This work seeks to further explore the polyionic coacervation fabrication process, and associated processing conditions under which protein encapsulation and subsequent release can be systematically and predictably manipulated so as to obtain desired effectiveness. BSA was used as a model protein which was encapsulated by either incorporation or incubation method, using the polyanion tripolyphosphate (TPP) as the coacervation crosslink agent to form chitosan-BSA-TPP nanoparticles. The BSA-loaded chitosan-TPP nanoparticles were characterized for particle size, morphology, zeta potential, BSA encapsulation efficiency, and subsequent release kinetics, which were found predominantly dependent on the factors of chitosan molecular weight, chitosan concentration, BSA loading concentration, and chitosan/TPP mass ratio. The BSA loaded nanoparticles prepared under varying conditions were in the size range of 200-580nm, and exhibit a high positive zeta potential. Detailed sequential time frame TEM imaging of morphological change of the BSA loaded particles showed a swelling and particle degradation process. Initial burst released due to surface protein desorption and diffusion from sublayers did not relate directly to change of particle size and shape, which was eminently apparent only after 6h. It is also notable that later stage particle degradation and disintegration did not yield a substantial follow-on release, as the remaining protein molecules, with adaptable 3-D conformation, could be tightly bound and entangled with the cationic chitosan chains. In general, this study demonstrated that the polyionic coacervation process for fabricating protein loaded chitosan nanoparticles offers simple preparation conditions and a clear processing window for manipulation of physiochemical properties of the nanoparticles (e.g., size and surface charge), which can be conditioned to exert control over protein encapsulation efficiency and subsequent release profile. The weakness of the chitosan nanoparticle system lies typically with difficulties in controlling initial burst effect in releasing large quantities of protein molecules.

Water-soluble chitosan nanoparticles as a novel carrier system for protein delivery

High MW chitosan (CS) solutions have already been proposed as vehicles for protein delivery. The aim of the present work is to investigate the potential utility of water-soluble chitosan (WSC) as vehicles to load and deliver proteins. WSC nanoparticles (WSC NP) with various formations were prepared based on ionic gelation of WSC with pentasodium tripolyphosphate (TPP) anions. Bovine serum albumin (BSA) was used as a model protein drug incorporated into the WSC nanoparticles. Blank and BSA-loaded WSC nanoparticles were examined and determined to have a spherical shape with diameters between 35–190 nm, and zeta potential between 35–42 mV. FTIR confirmed that the tripolyphosphoric groups of TPP linked to the ammonium groups of WSC in the nanoparticles. Some factors affecting delivery properties of BSA have been investigated. Altering the concentration of BSA from 0.05 to 1 mg/mL enhanced the loading capacity of BSA but decreased loading efficiency simultaneously. Also, with the introduction of poly ethylene glycol (PEG), BSA release accelerated. Nanoparticle preparation from WSC with various deacetylation degrees (DDs) from 72.6% to 90% and MWs ranging from 3.5 to 15.8 kDa promoted loading efficiency and decreased the release rate. These results indicate that WSC nanoparticles are promising carriers for protein delivery.

Carboxymethyl xanthan microparticles as a carrier for protein delivery

Xanthan gum (XG) was derivatized to sodium carboxymethyl xanthan gum (SCMXG) and then cross-linked with aluminium ions (Al+3) to prepare BSA-loaded microparticles (MPs) from a completely aqueous environment. The derivatized gum was characterized by various physical methods. Discrete and spherical BSA-loaded MPs were obtained from SCMXG solution, the pH of which was adjusted to 6 and 7 and the BSA entrapment efficiency was found to reach as high as 82%. The protein release in acidic dissolution medium was faster than that in alkaline dissolution medium and was accounted for the higher swelling ratio of the MPs in acidic environment. Moreover, the pH of the gum solution used to prepare the MPs also influenced the swelling and consequently protein release considerably.

Mannan‐mediated gene delivery for cancer immunotherapy

Recent years have seen a resurgence in interest in the development of efficient non-viral delivery systems for DNA vaccines and gene therapy. We have previously used oxidized and reduced mannan as carriers for protein delivery to antigen-presenting cells by targeting the receptors that bind mannose, resulting in efficient induction of cellular responses. In the present study, oxidized mannan and reduced mannan were used as receptor-mediated gene transfer ligands for cancer immunotherapy. In vivo studies in C57BL/6 mice showed that injection of DNA encoding ovalbumin (OVA) complexed to oxidized or reduced mannan-poly-L-lysine induced CD8 and CD4 T-cell responses as well as antibody responses leading to protection of mice from OVA+ tumours. Both oxidized and reduced mannan delivery was superior to DNA alone or DNA-poly-L-lysine. These studies demonstrate the potential of oxidized and reduced mannan for efficient receptor-mediated gene delivery in vivo, particularly as DNA vaccines for cancer immunotherapy.

Synthesis and Characterization of Insulin−Transferrin Conjugates

Receptor-mediated endocytosis can be exploited for improving the transcellular delivery of therapeutic proteins. Insulin conjugated to transferrin by forming disulfide bonds has been shown to improve insulin oral bioavailability in diabetic rats. We are developing a combination strategy involving complexation hydrogels as delivery vehicles for insulin-transferrin conjugates. The complexation hydrogels developed in our laboratory have been shown to be promising carriers for oral delivery of proteins and peptides. Integrating the strategies based on the complexation hydrogels and insulin-transferrin conjugates may prove to be a novel approach for oral delivery of insulin and other therapeutic proteins. In this work, electrospray ionization mass spectrometry (ESI-MS) was used to study the modification of insulin during its reaction with transferrin. The stability of the conjugated insulin to enzymatic degradation was also studied. ESI-MS studies confirmed the site-specific modifications of insulin. The transferrin conjugation of insulin was also shown to increase the stability of insulin to enzymatic degradation.

POLY(D, L- LACTIDE) NANOPARTICLES CONTAINING PROTEIN I. PREPARATION AND CHARACTERIZATION

For several years, large molecules such as peptides and proteins are not considered feasible candidates for controlled release systems since the physicochemical and biological properties of peptide and protein are different from those of conventional drugs. Therefore, specific delivery device for peptides and proteins needs to be developed. Previous literatures showed some advantages of polymeric nano- and micro-particles for the delivery of peptides and proteins to the systemic circulation and to immune system. The latest works further revealed the advantages of the nanoparticles over the microparticles. In this paper, we are reporting fabrication of poly (D, L- lactide) nanoparticles containing BSA protein by an improved double-emulsion (water-in-oil-in-water) solvent evaporation /extraction technique. The BSA-loaded PDLLA nanoparticles are characterized in terms of size, zeta potential, morphology and BSA-loaded encapsulation efficiency, using various analytical techniques such as HR-TEM, laser light scattering method, FT-IR and micro-BCA assay. Results show that the encapsulation efficiency is 53.3%, particle size about 350 nm and zeta potential –48.18 mv. IR spectra suggest the absence of strong chemical interactions between BSA and PDLLA and the physicochemical integrity of BSA is retained within the nanoparticles. The current study suggests that the PDLLA nanoparticles could be an effective carrier for protein and peptide delivery. Based on the results above, we are expecting to construct a preparation fundamental for sensitive bio-macromolecules nano-delivery system.

Controlled release of bovine serum albumin using MPEG–PCL diblock copolymers as implantable protein carriers

MPEG–PCL diblock copolymers consisting of methoxy polyethylene glycol (MPEG) and poly(ϵ-caprolactone) (PCL) as drug carriers were synthesized by ring-opening polymerization. It is possible to control the balance between hydrophilic and hydrophobic by changing the MPEG and the ratio of ϵ-CL to MPEG. Implantable wafers were easily fabricated by the direct compression method after physical mixing of diblock copolymers and bovine serum albumin–fluorescein isothiocyanate (BSA-FITC) as a model protein drug. The BSA release from wafers prepared by MPEG–PCL diblock copolymers were higher than that from PCL with the physical blending of MPEG. The wafers prepared by a variety of MPEG–PCL diblock copolymers exhibited the controlled BSA release profiles with a dependence on MPEG–PCL diblock copolymer compositions. In addition, the changing of MPEG and PCL molecular weights within MPEG–PCL diblock copolymer controlled the initial burst of BSA. We confirmed that the diblock copolymers could be served as protein delivery carrier in implantable wafer form. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102:1561–1567, 2006

Fucosphere—New microsphere carriers for peptide and protein delivery: Preparation and in vitro characterization

Purpose: Fucoidan is a complex polysaccharide containing sugars and high amounts of sulphate derived from marine brown algaes. In this study, a new microsphere-delivery system based on cross-linking of fucoidan with chitosan, named Fucosphere, was evaluated as a drug carrier. Bovine serum albumin (BSA) was used as a model protein. The effect of fucoidan (1.5, 1.75, 2.0 and 2.5%), chitosan (0.25, 0.50 and 0.75%) and protein (0.25, 0.50 and 0.75%) concentrations, the origin of chitosan and the preparation methods of the particles on the microsphere characteristics were evaluated.Methods: The microspheres were prepared by a simple method based on the cross-linking of the opposite charged biopolymers. The shape and surface morphologies of the particles were evaluated by scanning electron microscopy (SEM) and the size, charge and encapsulation capacity of the microspheres were determined. The released amount of BSA from the microspheres into phosphate buffered saline (PBS pH 7.4) was determined spectrophotometrically by the Bradford method. SDS-PAGE was performed to check the structural integrity of BSA after the preparation.Results: Smooth and spherical microspheres between the size ranges of 0.61–1.28 µm were obtained. BSA was efficiently encapsulated into the microspheres (51.8–89.5%). All formulation parameters affected the encapsulation capacity of Fucosphere (p < 0.05). The highest encapsulation was obtained with microspheres containing 2.5% of fucoidan (89.5%).Conclusions: The extent of drug release from the microspheres was dependent on the concentrations of polymers and BSA, chitosan origin and type of preparation method. When the addition methods of protein compared, BSA encapsulated into Fucosphere released slower than the adsorbed protein (E) (p < 0.05). The electrophoretic mobility values of Fucospheres changed between +6.9 and +32.3 mV. In general, BSA release from Fucosphere showed a three-phasic release curve. In conclusion, this new fucoidan microsphere system may be a potential delivery of macromolecular drug such as peptide and protein.

Translocation of positively charged copoly(Lys/Tyr) across phospholipid membranes

Much attention has recently been paid to the study of positively charged polypeptides as a possible carrier for therapeutic protein or DNA delivery to cells. In this study, we have investigated the translocation of positively charged copoly(Lys/Tyr) (MW = 72000, DP = 385) across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipids (SBPL) using ζ potential method, circular dichroism spectroscopy (CD), electrophysiology technique, fluorescence spectroscopy, and confocal laser scanning microscopy. Results of ζ potentials show that copoly(Lys/Tyr) associate with lipid membranes and become gradually saturated on the membranes either hydrophobically or electrostatically or both. CD studies demonstrate that the copoly(Lys/Tyr) takes and remains β-sheet conformation during its interaction with liposome membranes, indicating that the translocation process should be carpet-mode like. Data from the electrophysiology technique reveal that positively charged copoly(Lys/Tyr) can cause transmembrane currents under an applied voltage, confirming its transfer across lipid membranes. Fluorescence spectroscopy results display a three-step mechanism of translocation across membrane: adsorption, transportation, and desorption, which has been verified by results from confocal laser scanning microscopy. We provided the first direct observation that the positively charged polypeptides, copoly(Lys/Tyr), can translocate through SBPL and EPC/DOPE lipid bilayer membranes. In addition, we found that the translocation efficiency of copoly(Lys/Tyr) was higher on the EPC/DOPE lipid membrane than on the SBPL lipid membrane.