Protein Entrapment Efficiency Research Articles

Triple growth factor delivery promotes functional bone regeneration following composite musculoskeletal trauma

Successful bone healing in severe trauma depends on early revascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote revascularization following severe bone injuries; however, results have been inconsistent. This is the first study investigating the effects of dual angiogenic growth factors (VEGF and PDGF) with low-dose bone morphogenetic protein-2 (BMP-2; 2.5 µg) on bone healing in a clinically challenging composite bone-muscle injury model. Our hydrogel-based delivery systems demonstrated a more than 90% protein entrapment efficiency and a controlled simultaneous release of three growth factors over 28 days. Co-stimulation of microvascular fragment constructs with VEGF and PDGF promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined VEGF (5 µg) and PDGF (7.5 µg or 15 µg) delivery with a low dose of BMP-2 significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. Notably, the regenerated bone mechanics reached ~60% of intact bone, a value that was previously only achieved by delivery of high-dose BMP-2 (10 µg) in this injury model. Overall, sustained delivery of VEGF, PDFG, and BMP-2 is a promising strategy to promote functional vascularized bone tissue regeneration following severe composite musculoskeletal injury. Although this study is conducted in a clinically relevant composite injury model in rats using a simultaneous release strategy, future studies are necessary to test the regenerative potential of spatiotemporally controlled delivery of triple growth factors on bone healing using large animal models. Statement of significanceVolumetric muscle loss combined with delayed union or non-union bone defect causes deleterious effects on bone regeneration even with the supplementation of bone morphogenetic protein-2 (BMP-2). In this study, the controlled delivery of dual angiogenic growth factors (vascular endothelial growth factor [VEGF] + Platelet-derived growth factor [PDGF]) increases vascular growth in vitro. Co-delivering VEGF+PDGF significantly increase the bone formation efficacy of low-dose BMP-2 and improves the mechanics of regenerated bone in a challenging composite bone-muscle injury model.

Poly (Lactic-co-Glycolic Acid) Nanoparticles and Nanoliposomes for Protein Delivery in Targeted Therapy: A Comparative In Vitro Study.

Over the previous years, the design, development, and potential application of nanocarriers in the medical field have been intensively studied for their ability to preserve drug properties, especially their pharmacological activity, and to improve their bioavailability. This work is a comparative study between two different types of nanocarriers, poly (lactic-co-glycolic acid)-based nanoparticles and phosphatidylcholine-based nanoliposomes, both prepared for the encapsulation of bovine serum albumin as a model protein. Polymeric nanoparticles were produced using the double emulsion water-oil-water evaporation method, whereas nanoliposomes were obtained by the thin-film hydration method. Both nanocarriers were characterized by morphological analysis, particle mean size, particle size distribution, and protein entrapment efficiency. In vitro release studies were performed for 12 days at 37 °C. In order to explore a possible application of these nanocarriers for a targeted therapy in the cardiovascular field, hemolytic activity and biocompatibility, in terms of cell viability, were performed by using human red blood cells and EA.hy926 human endothelial cell line, respectively.

Ion-exchanging dialysis as an effective method for protein entrapment in curdlan hydrogel.

The purpose of this study was to determine whether dialysis method allows for efficient protein entrapment in curdlan-based hydrogel. Thus, bovine serum albumin, a model of bioactive protein, was incorporated into curdlan matrix using ion-exchanging dialysis method against two concentrations of CaCl2 solution - 2% and 10%, respectively. Then, physicochemical, mechanical, and biological properties of the bovine serum albumin-loaded curdlan hydrogels were evaluated. Received results show that neither the polymer nor the entrapment procedure change the bovine serum albumin conformation (as proven by Fourier transform infrared spectroscopy and circular dichroism spectroscopy) and the process guarantees high protein entrapment efficiency (above 95%). The curdlan-based carrier obtained against 2% of CaCl2 solution was found to possess higher swelling ability, release greater amounts of bovine serum albumin (up to 4 weeks), and exhibit superior biocompatibility compared to curdlan-based carrier obtained against 10% of CaCl2 solution. Thus, dialysis method enables efficient protein entrapment in curdlan hydrogel and obtained protein carrier via dialysis method into 2% of CaCl2 solution may be considered as a promising protein delivery system especially for tissue engineering applications. It should be noted that we are the first who presented effective method for protein entrapment in curdlan hydrogel.

Development of bi-polymer lipid hybrid nanocarrier (BLN) to improve the entrapment and stability of insulin for efficient oral delivery

Solid lipid nanoparticles (SLN) have demonstrated favorable properties for oral protein delivery. However, their protein entrapment efficiency (EE%) values remain limited due to their hydrophobic nature. In this study, we reported a new strategy in which two polymeric excipients were incorporated into the double-emulsion based SLN to address this entrapment inefficiency issue. Using insulin as the model protein drug, the resulting hybrid nanocarriers known as bi-polymer lipid nanocarriers (BLN) were evaluated for physicochemical properties, drug and particle stability, in vitro biological behaviors and in vivo hypoglycemic effect. It was found that BLN containing PEG 6000 in internal aqueous phase and PLGA in lipid phase showed favorable size (around 240 nm) and reasonably narrow particle size distribution. The bi-polymer strategy improved insulin EE% over standard w/o/w SLN by 2.5-fold (∼50% versus 20%) while preserving the insulin chemical stability and biological activity. BLN demonstrated good dispersion stability under gastrointestinal conditions, protected the entrapped insulin against enzymatic degradation well, and were well internalized into Caco-2 cells with minimal cytotoxicity. Pharmacological availability of oral insulin delivered by BLN (6.1%) was comparable to lectin-modified SLN. To conclude, BLN is a promising hybrid nanocarrier design to achieve efficient oral insulin delivery.

Optimizing liposomes for delivery of Bowman-Birk protease inhibitors - Platforms for multiple biomedical applications.

One of the major challenges in the administration of therapeutic proteins involves delivery limitations. Liposomes are well-known drug delivery systems (DDS) that have been used to overcome this drawback; nevertheless, low protein entrapment efficiency (EE) still limits their wide biomedical application on a commercial scale. In the present work, different methods for protein entrapment into liposomes were tested in order to obtain tailored DDS platforms for multiple biomedical applications. The protein used as model was the Black-eyed pea Trypsin and Chymotrypsin Inhibitor (BTCI), a member of the Bowman-Birk protease inhibitor family (BBIs), which has been largely explored for its potential application in many biomedical therapies. We optimized reverse-phase evaporation (REV) and freeze/thaw (F/T) entrapment methods, using a cationic lipid matrix to entrap expressive amounts of BTCI (∼100 μM) in stable liposomes without affecting its protease inhibition activity. The influence of various parameters (e.g. entrapment method, liposome composition, buffer type) on particle size, charge, polydispersity, and EE of liposomes was investigated to provide an insight on how to control such parameters in view of obtaining a high entrapment yield. In addition, BTCI liposome platforms obtained herein showed to be versatile vesicles, allowing surface modification with moieties/polymers of interest (e.g. PEG, transferrin). The aforementioned results are relevant to focusing on the entrapment of other promising BBIs or protein agents sharing similar structural features. These findings encourage future studies to investigate the advantages of using the liposome platforms presented herein to broaden the use of this type of DDS for BBI biomedical applications.

Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres.

Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

Combined Poly(Lactide-Co-Glycolide) Microspheres Containing Diphtheria Toxoid for a Single-shot Immunization.

To develop a single-shot vaccine containing diphtheria toxoid (DT) with a sufficient immune response, poly(lactide-co-glycolide) (PLGA) microspheres were prepared by water-in-oil-in-water double emulsification and solvent extraction techniques using low or high-molecular-weight PLGA (LMW-MS or HMW-MS). Stearic acid (SA) was introduced to HMW-MS (HMW/SA-MS) as a release modulator. Mean particle sizes (dvs, μm) varied between the prepared microspheres, with LMW-MS, HMW-MS, and HMW/SA-MS having the sizes of 29.83, 110.59, and 69.5μm, respectively; however, the protein entrapment and loading efficiency did not vary, with values of 15.2-16.8μg/mg and 61-75%, respectively. LMW-MS showed slower initial release (~ 2weeks) but faster and higher release of antigen during weeks 3~7 than did HMW-MS. HMW/SA-MS showed rapid initial release followed by a continuous release over an extended period of time (~ 12weeks). Mixed PLGA microspheres (MIX-MS), a combination of HMW/SA-MS and LMW-MS (1:1), demonstrated a sufficient initial antigen release and a subsequent boost release in a pulsatile manner. Serum antibody levels were measured by ELISA after DT immunization of Balb/c mice, and showed a greater response to MIX-MS than to alum-adsorbed DT (control). A lethal toxin challenge test with MIX-MS (a DT dose of 18Lf) using Balb/c mice revealed complete protection, indicating a good candidate delivery system for a single-shot immunization.

Structure and properties of hybrid biopolymer particles fabricated by co-precipitation cross-linking dissolution procedure

The Co-precipitation Crosslinking Dissolution technique (CCD-technique) allows a few-steps fabrication of particles composed of different biopolymers and bioactive agents under mild conditions. Morphology and properties of the fabricated biopolymer particles depend on the fabrication conditions, the nature of the biopolymers and additives, but also on the choice of the inorganic templates for co-precipitation. Here, we investigate the influence of an acidic biopolymer, hyaluronic acid (HA), on the formation of particles from bovine hemoglobin and bovine serum albumin applying co-precipitation with CaCO3 and MnCO3. CaCO3 templated biopolymer particles are almost spherical with particle size from 2 to 20 µm and protein entrapment efficiency from 13 to 77%. Presence of HA causes significant structural changes of the particles and decreasing protein entrapment efficiency. In contrast, MnCO3 templated particles exhibit uniform peanut shape and submicron size with remarkably high protein entrapment efficiency of nearly 100%. Addition of HA has no influence on the protein entrapment efficiency or on morphology and size of the particles. These effects can be attributed to the strong interaction of Mn2+ with proteins and much weaker interaction with HA. Therefore, entrapment efficiency, size and structure of biopolymer particles can be optimized by varying the mineral templates and additives.

Entrapment of protein in chitosan-tripolyphosphate beads and its release in an in vitro digestive model.

This research sought to evaluate the entrapment and in vitro release behaviour of bovine serum albumin (BSA) in chitosan-tripolyphosphate (TPP) hydrogel beads. Beads were manufactured by extruding gel forming solutions containing varying concentrations of chitosan (1-2.5%w/w) and BSA (0.25-10%w/w) into TPP solutions ranging in concentration from 0.1 to 10%w/w and in ionic strength from 0.16 to 0.67M at pH values of 4, 5 and 9.4. Beads produced at a low TPP concentration of 0.4% w/w had the highest BSA entrapment efficiency (71.6±0.7%) and inhibited BSA release in simulated gastric fluid (SGF) to a greater extent. Increasing chitosan concentration resulted in a higher protein entrapment efficiency, but lowered the overall release. Increasing TPP concentration or the BSA concentration loaded, led to early release in SGF. The results indicate that the utilization of lower concentrations of TPP is a good approach to improve the protein retention ability of chitosan-TPP beads in a simulated gastric environment.

Growth Factor-Loaded Nano-niosomal Gel Formulation and Characterization.

Controlled delivery of signaling factors could be a great approach in the tissue engineering field. Nano-niosomal drug delivery systems offer numerous advantages for this purpose. The present study reports the formulation and evaluation of a growth factor (GF)-loaded nano-niosome-hydrogel composite for GF delivery to modulate cell behavior. Niosomes were prepared, using span 60 surfactant with cholesterol (CH) in diethyl ether solvent, by reverse-phase evaporation technique. Basic fibroblast growth factor (bFGF) and bovine serum albumin (BSA) were loaded simultaneously and the final suspension was embedded into agarose hydrogel. Particle size, vesicle morphology, protein entrapment efficiency (EE), and release profile were measured by dynamic light scattering (DLS) nanoparticle size analyzer, transmission electron microscopy (TEM) and NanoDrop spectrophotometry methods, respectively. The release and performance of bFGF were revealed via human umbilical vein endothelial cell (HUVEC) proliferation using microscopy imaging and MTT assay. Nano-niosomes had an average particle size of 232nm and had encapsulated 58% of the total proteins present in the suspension. bFGF-BSA-loaded niosomal gel considerably enhanced HUVEC proliferation. This GF-loaded niosomal hydrogel could be a potent material in many biomedical applications including the induction of angiogenesis in tissue engineering.

In vitro characterisation of Span™ 65 niosomal formulations containing proteins

Proteins can be encapsulated in niosomes as they are known to protect proteins against the surrounding environment. Niosomes of Span 65/cholesterol/pluronic F -127 were prepared by thin film hydration method. Insulin and lysozyme were chosen as model proteins. Niosomes were characterised for morphology by Transmission Electron Microscopy (TEM) and vesicles size using Dynamic Light Scattering. The entrapment efficiency of protein in niosomes was determined by complete vesicle disruption using 50:50% isopropanol:buf fer, followed by analysis of the resulting solutions by HPLC method. Thermal behaviour of the niosomes was investigated using Differential Scanning Calorimetry (DSC). Protection of proteins against simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were also assessed. The results showed that niosomes prepared with different molar ratios % of Span 65, cholesterol and pluronic F-127 successfully produced with insulin and lysozyme. For insulin containing niosomes, the ratio % of 64.7 (Span 65): 32.3 (cholesterol): 3.0 (pluronic F - 127) produced the highest protein encapsulation efficiency and the smallest vesicle size of 653.6 nm. For lysozyme containing niosomes, the maximum protein encapsulation was found in 72.75/24.25/3.00% molar ratio of Span 65/cholesterol/pluronic F -127 niosomes with vesicle size of 627.3 nm. The release study of proteins from the niosomal preparations in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) revealed that insulin and lysozyme efflux from the niosomes is a biphasic process. The results indicate that Span 65 niosomes could be developed as controlled release dosage forms for delivery of peptides and proteins such as insulin and lysozyme.

Preparation of microspheres of superoxide dismutase and their activities

To prepare the poly(lactic-co-glycolic acid) (PLGA) microspheres and composite alginate-chitosan-PLGA microspheres containing superoxide dismutase (SOD) and to evaluate their SOD activities. The SOD-PLGA microspheres were prepared by W/O/W emulsification method, and the composite microspheres were prepared by two steps:alginate-chitosan microcapsules were first prepared by a modified emulsification and ion crosslinking method, and then they were further dispersed in PLGA to form the composite microspheres. The SOD concentration was determined by Coomassie method, its activity was measured by xanthine oxidase system. The SOD activity was less sensitive to temperature and sensitive to pH, organic solvents, ultrasound and vigorous stir without iced bath. The entrapment efficiencies of SOD in PLGA (50:50) microspheres, PLGA (70:30) microspheres, alginate-chitosan microcapsules, the composite PLGA (50:50) microspheres and the composite PLGA (70:30) microspheres were 36.42%±1.81%, 66.18%±0.05%, 91.08%±1.28%, 87.30%±3.89% and 83.19%±3.48%, respectively. In vitro release tests demonstrated that the SOD activities in 50:50 composite microspheres were higher than that in the PLGA ones at 1 h, 8 h and 1 w. The composite alginate-chitosan-PLGA microspheres for SOD sustained release can significantly improve the protein entrapment efficiency and maintain its protein activity.

Alginate/BSA/montmorillonite composites with enhanced protein entrapment and controlled release efficiency

Novel pH sensitive alginate–protein–clay composite beads were investigated for the in vitro oral delivery of the model protein, bovine serum albumin (BSA). X-ray diffraction (XRD) results revealed that BSA enter between layers of montmorillonite (MMT) by expanding interlayer distance and finally an exfoliated structure forms in the alginate hydrogel. MMT incorporation increases protein entrapment efficiency to 78%, compared to 40% of conventional alginate beads. The release ratio of BSA from composite beads is 9–13% depending on MMT contents after around a 2h stay in gastric fluid. More importantly, no BSA release is detected until 60–90min after the first contact time of beads with gastric solution. The presence of clay in alginate beads prevents burst release in higher pH of intestine by slowing release rate of BSA to 45–55% within around 9h, resulting in a potential matrix for intestinal release of protein drugs.

Spray-Dried Chitosan Microparticles for Cellular Delivery of an Antigenic Protein: Physico-chemical Properties and Cellular Uptake by Dendritic Cells and Macrophages

Spray-dried chitosan microparticles for cellular delivery of antigen to dendritic cells (DC) and macrophages (Mϕ) were investigated. Chitosan microparticles were prepared by spray drying. For comparison, poly(lactic-co-glycolic acid) (PLGA) and poly(α-butyl cyanoacrylate) (BCA) micro-/nanoparticles were generated. Bovine serum albumin (BSA) was used as a model antigen. The particles were characterized in terms of size, morphology, surface charge, surface composition, protein content, entrapment efficiency, in vitro release, and protein integrity. Additionally, they were subject to cell viability and cellular uptake study with DC and Mϕ. Size of chitosan, PLGA, and BCA micro-/nanoparticles ranged between 3.11-7.18, 0.94-6.26, and 0.30-6.34μm, respectively. Particle morphology and in vitro protein release varied, depending on polymer type, particle composition and preparation process parameters. Chitosan microparticles were cationic, while PLGA microparticles were neutral. BCA micro-/nanoparticles were either anionic or cationic, according to polymerization pH. Protein content and entrapment efficiency of chitosan and PLGA microparticles were relatively consistent. Only integrity and conformational structure of protein encapsulated in chitosan microparticles were completely retained. Chitosan and PLGA microparticles were non-toxic to DC and Mϕ, but the former were internalized more efficiently. Spray-dried chitosan microparticles delivered the antigen efficiently to DC and Mϕ.

Polymeric particles for the controlled release of human amylin

Since its discovery the therapeutic use of the pancreatic hormone amylin has been limited due to its poor water solubility and propensity for amyloid aggregation. We have entrapped the human amylin protein in polymeric nanoparticles, using a single emulsion–solvent evaporation method and investigated its effectiveness in the controlled release of the peptide. Typical preparations composed of poly-ɛ-caprolactone had a mean particle size of approximately 200nm, low polydispersity index, high protein entrapment efficiency (80%) and process yield (90%), and spherical and smooth surfaces. These nanoparticles presented a controlled release in vitro for approximately 240h. Pharmacological evaluation in vivo by subcutaneous administration in fasting mice demonstrated the bioactivity and effectiveness of the released human amylin, resulting in reduced glycemia lasting for at least 36h. These features indicate the potential for the use of a confined particulate system in the therapeutic controlled and sustained release of human amylin.

Preparation of Chitosan Nanoparticles for Protein Delivery by w/o/w Emulsion Solvent Evaporation and Simple Ionotropic Gelation Techniques

The purpose of this study was to prepare chitosan nanoparticles (CS NP) for controlled protein delivery. Two techniques, simple ionotropic gelation (method [I]) and w/o/w emulsion solvent evaporation containing ionotropic gelation (method [II]), were used to prepare CS NP. Tripolyphosphate (TPP) and Eudragit L100-55 (Eud) were used as anionic agents to form complex with cationic chitosan. Bovine serum albumin (BSA) was encapsulated into NP. The morphological characteristics, particle size and size distribution, protein entrapment efficiency, zeta potential, in vitro release, protein secondary structure and its integrity were investigated. The results showed that CS NP could be prepared by appropriate cationic and anionic ratios in both methods. Excess anionic agents resulted in particle aggregation of micron size. The median sizes of particles were between 0.127-0.273 mcm with method [I] provided the smallest size. The 0.02-0.10% BSA loaded preparations showed the same particle sizes and size distributions as blank preparations. SEM photomicrographs revealed that the obtained NP were spherical. Protein entrapment efficiency was between 47-84% and increased when decreasing the percentage of drug loading. The method [II] with TPP exhibited the highest protein entrapment efficiency, following by the method [II] with Eud and method [I] with TPP, respectively. The zeta potentials were positive. Prolonged in vitro protein release profiles were observed from all preparations of CS NP. After 10 days, the release was between 53-72%. Circular dichroism and SDS-polyaceylamide gel electrophoresis techniques confirmed that these processes did not have any destructive effect on the protein structure. Therefore these preparation techniques could be used to encapsulate water-soluble drugs, proteins, DNA, or antigens into CS NP as effective delivery carriers.

A protein delivery system: biodegradable alginate–chitosan–poly(lactic-co-glycolic acid) composite microspheres

In the present study we developed alginate–chitosan–poly(lactic-co-glycolic acid) (PLGA) composite microspheres to elevate protein entrapment efficiency and decrease its burst release. Bovine serum albumin (BSA), which used as the model protein, was entrapped into the alginate microcapsules by a modified emulsification method in an isopropyl alcohol-washed way. The rapid drug releases were sustained by chitosan coating. To obtain the desired release properties, the alginate–chitosan microcapsules were further incorporated in the PLGA to form the composite microspheres. The average diameter of the composite microcapsules was 31 ± 9 μm and the encapsulation efficiency was 81–87%, while that of conventional PLGA microspheres was just 61–65%. Furthermore, the burst releases at 1 h of BSA entrapped in composite microspheres which containing PLGA (50:50) and PLGA (70:30) decreased to 24% and 8% in PBS, and further decreased to 5% and 2% in saline. On the contrary, the burst releases of conventional PLGA microspheres were 48% and 52% in PBS, respectively. Moreover, the release profiles could be manipulated by regulating the ratios of poly(lactic acid) to poly(glycolic acid) in the composite microspheres.

Preparation of poly[dl-lactide-co-glycolide]-based microspheres containing protein by use of amphiphilic diblock copolymers of depsipeptide and lactide having ionic pendant groups as biodegradable surfactants by W/O/W emulsion method

To develop the preparative method for poly(dl-lactide-co-glycolide)-based microspheres containing proteins, we prepared microspheres from mixture of poly(dl-lactide-co-glycolide) and polydepsipeptide-block-poly(dl-lactide) having cationic or anionic pendant groups. Since the latter amphiphilic copolymers consisting of hydrophobic poly(dl-lactide) segment and hydrophilic polydepsipeptide segment with amino or carboxyl groups could be converted to cationic or anionic block copolymers, they could act as biodegradable surfactants on the preparation of poly(dl-lactide-co-glycolide)-based microspheres by water-in-oil-in-water emulsion method. The amphiphilic block copolymers were established to stabilize primary emulsions on the preparation of microspheres by scanning electron microscopy. We investigated the effects of the addition of the block copolymers on the entrapment efficiency of protein, the release behavior of protein from microspheres and the stability of protein.

Encapsulating protein into preformed liposomes by ethanol-destabilized method.

This study describes a highly efficient method for encapsulating protein drugs into liposomes without using toxic solvents such as chloroform. Large unilamellar vesicles (LUVs) were formed by the ethanol injection method. The effects of composition of phospholipid, buffer concentration, incubation time, incubation temperature, drug loading, ethanol content, and the presence of poly(ethylene) glycol (PEG) lipids on the entrapment efficiency of protein were investigated. It was shown that these preformed LUVs could be induced to entrap protein drugs in the presence of ethanol. Protein could be efficiently encapsulated into liposomes. The interaction of the liposomes with proteins leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. Protein drugs were stable in the liposomal solution. There is no significant activity loss during the encapsulation process. The optimal encapsulation efficiency was achieved when 30% approximately 40% ethanol was used in encapsulating protein drugs. Due to the steric hindrance, LUVs containing a PEG coating will dramatically reduce the encapsulation efficiency, even in liposomes containing very low amount of PEG.

Polyanion/gelatin complexes as pH‐sensitive gels for controlled protein release

AbstractPolyanion/gelatin complexes including poly(methacrylic acid) (PMAA)/gelatin, poly(acrylic acid) (PAA)/gelatin, and heparin/gelatin are investigated as pH‐sensitive gels for controlled protein release. Polyanions can interact with gelatin and form amorphous precipitates within a certain pH range, which is affected by the polyanion nature. The entrapment efficiency of model proteins (myoglobin, cytochrome c, and pepsin) into the complexes is rather high (>80%). By using a modified colloid titration that mixes a solution of gelatin and model proteins titrated with polyanion solution, myoglobin and cytochrome c are found to interact with polyanions by electrostatic forces at low pH, while pepsin either interacts with the polyanion when the pH is below its isoelectric point (IEP) or complexes with gelatin at a pH above IEPpepsin. At pH 7.4 all the complexes dissociate and proteins are rapidly released within a few hours. The complexes are stable and the proteins are retained within a certain pH range, which is related to the polyanion type (e.g., 5.0–2.0 for PMAA, 4.6–1.2 for PAA, and <4.3 for heparin). The three processes of complex formation, dissociation, and protein release have a good correlation. In addition, the protein release transition takes place within a rather narrow pH range (ca. 0.5 units) and the protein nature has little effect on the protein release profile. The high protein entrapment efficiency and good pH sensitivity of the protein release can be mainly attributed to the electrostatic attractive interactions between proteins and polyanion or gelatin. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 1416–1425, 2001