Decoration of silk fibroin by click chemistry for biomedical application.

Silkfibroin (SF) has an excellent biocompatibility and its remarkable structure translates into exciting mechanical properties rendering this biomaterial particularly fascinating for biomedical application. To further boost the material's biological/preclinical impact, SF is decorated with biologics, typically by carbodiimide/N-hydroxysuccinimide coupling (EDC/NHS). For biomedical application, this chemistry challenges the product risk profile due to the formation of covalent aggregates, particularly when decoration is with biologics occurring naturally in humans as these aggregates may prime for autoimmunity. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry) provides the necessary specificity to avoid such intermolecular, covalent aggregates. We present a blueprint outlining the necessary chemistry rendering SF compatible with CuAAC and with a particular focus on structural consequences. For that, the number of SF carboxyl groups (carboxyl-SF; required for EDC/NHS chemistry) or azido groups (azido-SF; required for click chemistry) was tailored by means of diazonium coupling of the SF tyrosine residues. Structural impact on SF and decorated SF was characterized by Fourier transform infrared spectroscopy (FTIR). The click chemistry yielded a better controlled product as compared to the EDC/NHS chemistry with no formation of inter- and intramolecular crosslinks as demonstrated for SF decorated with fluorescent model compounds or a biologic, fibroblast growth factor 2 (FGF2), respectively. In conclusion, SF can readily be translated into a scaffold compatible with click chemistry yielding decorated products with a better risk profile for biomedical application.

Controlled protein delivery from electrospun non-wovens: novel combination of protein crystals and a biodegradable release matrix.

Poly-ε-caprolactone (PCL) is an excellent polymer for electrospinning and matrix-controlled drug delivery combining optimal processability and good biocompatibility. Electrospinning of proteins has been shown to be challenging via the use of organic solvents, frequently resulting in protein unfolding or aggregation. Encapsulation of protein crystals represents an attractive but largely unexplored alternative to established protein encapsulation techniques because of increased thermodynamic stability and improved solvent resistance of the crystalline state. We herein explore the electrospinning of protein crystal suspensions and establish basic design principles for this novel type of protein delivery system. PCL was deployed as a matrix, and lysozyme was used as a crystallizing model protein. By rational combination of lysozyme crystals 0.7 or 2.1 µm in diameter and a PCL fiber diameter between 1.6 and 10 µm, release within the first 24 h could be varied between approximately 10 and 100%. Lysozyme loading of PCL microfibers between 0.5 and 5% was achieved without affecting processability. While relative release was unaffected by loading percentage, the amount of lysozyme released could be tailored. PCL was blended with poly(ethylene glycol) and poly(lactic-co-glycolic acid) to further modify the release rate. Under optimized conditions, an almost constant lysozyme release over 11 weeks was achieved.

Enzymatic degradation pattern of polysorbate 20 impacts interfacial properties of monoclonal antibody formulations.

Polysorbate 20 (PS20) is widely used to maintain protein stability in biopharmaceutical formulations. However, PS20 is susceptible to hydrolytic degradation catalyzed by trace amounts of residual host cell proteins present in monoclonal antibody (mAb) formulations. The resulting loss of intact surfactant and the presence of PS20 degradation products, such as free fatty acids (FFAs), may impair protein stability. In this study, two hydrolytically-active immobilized lipases, which primarily targeted either monoester or higher-order ester species in PS20, were used to generate partially-degraded PS20. The impact of PS20 degradation pattern on critical micelle concentration (CMC), surface tension, interfacial rheology parameters and agitation protection was assessed. CMC was slightly increased upon monoester degradation, but significantly increased upon higher-order ester degradation. The PS20 degradation pattern also significantly impacted the dynamic surface tension of a mAb formulation, whereas changes in the equilibrium surface tension were mainly caused by the adsorption of FFAs onto the air-water interface. In an agitation protection study, monoester degradation resulted in the formation of soluble mAb aggregates and proteinaceous particles, suggesting that preferential degradation of PS20 monoester species can significantly impair mAb stability. Additional mAbs should be tested in the future to assess the impact of the protein format.

Comparison of Protein-like Model Particles Fabricated by Micro 3D Printing to Established Standard Particles.

Innovative analytical instruments and development of new methods has provided a better understanding of protein particle formation in biopharmaceuticals but have also challenged the ability to obtain reproducible and reliable measurements. The need for protein-like particle standards mimicking the irregular shape, translucent nature and near-to-neutral buoyancy of protein particles remained one of the hot topics in the field of particle detection and characterization in biopharmaceutical formulations. An innovative protein-like particle model has been developed using two photo polymerization (2PP) printing allowing to fabricate irregularly shaped particles with similar properties as protein particles at precise size of 50 µm and 150 µm, representative of subvisible particles and visible particles, respectively. A study was conducted to compare the morphological, physical, and optical properties of artificially generated protein particles, polystyrene spheres, ETFE, and SU-8 particle standards, along with newly developed protein-like model particles manufactured using 2PP printing. Our results suggest that 2PP printing can be used to produce protein-like particle standards that might facilitate harmonization and standardization of subvisible and visible protein particle characterization across laboratories and organizations.

Not the Usual Suspects: Alternative Surfactants for Biopharmaceuticals.

Therapeutically relevant proteins naturally adsorb to interfaces, causing aggregation which in turn potentially leads to numerous adverse consequences such as loss of activity or unwanted immunogenic reactions. Surfactants are ubiquitously used in biotherapeutics drug development to oppose interfacial stress, yet, the choice of the surfactant is extremely limited: to date, only polysorbates (PS20/80) and poloxamer 188 are used in commercial products. However, both surfactant families suffer from severe degradation and impurities of the raw material, which frequently increases the risk of particle generation, chemical protein degradation, and potential adverse immune reactions. Herein, we assessed a total of 40 suitable alternative surfactant candidates and subsequently performed a selection through a three-gate screening process employing four protein modalities encompassing six different formulations. The screening is based on short-term agitation-induced aggregation studies coupled to particle analysis and surface tension characterization, followed by long-term quiescence stability studies connected to protein purity measurements and particle analysis. The study concludes by assessing the surfactant's chemical and enzymatic degradation propensity. The candidates emerging from the screening are de novo α-tocopherol-derivatives named VEDG-2.2 and VEDS, produced ad hoc for this study. They display protein stabilization potential comparable or better than polysorbates together with an increased resistance to chemical and enzymatic degradation, thus representing valuable alternative surfactants for biotherapeutics.

Integrin alphaVbeta3 targeted gene delivery using RGD peptidomimetic conjugates with copolymers of PEGylated poly(ethylene imine).

This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.

Influence of morphology and drug distribution on the release process of FITC-dextran-loaded microspheres prepared with different types of PLGA.

The aim of the present work was to understand the collaborative roles and the comprehensive effects of polymer nature, morphology, drug distribution and release behaviour for PLGA microspheres prepared by the double emulsion method. The morphology and drug distribution of the FITC-dextran-loaded microspheres were investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), respectively. The results show that the morphology and release profiles depend on the polymer nature. For the capped PLGA RG502, the porosity, pore size and drug distribution had no pronounced influence on the release profile beyond the initial release. No significant changes in size and morphology were found and there was negligible water uptake during the release process. PEG addition as a pore maker indicated a possible way to modify the release rate at the second release stage. However, in the case of the uncapped PLGA RG503H, release profiles were dependent upon changes in porosity, pore size and drug loading. Increases in porosity, internal pore size and loading resulted in a continuous release profile. Previous studies have shown the importance of different process parameters on morphology and drug release, but in this work it is clear that polymer nature is a determining factor.

A Versatile Biocompatible Antibiotic Delivery System Based on Self-Assembling Peptides with Antimicrobial and Regenerative Potential.

Periodontitis is a chronic inflammatory and tissue-destructive disease. Since the polymicrobiome in the oral cavity makes it difficult to treat, novel therapeutic strategies are required. Hydrogels based on self-assembling peptides (SAP) can be suitable candidates for periodontal therapy due to their injectability, biocompatibility, cargo-loading capacity, and tunable physicochemical and mechanical properties. In this study, two SAP hydrogels (P11-4 and P11-28/29) are examined for their intrinsic antimicrobial activity, regenerative potential, and antibiotic delivery capacity. A significant antibacterial effect of P11-28/29 hydrogels on the periodontal pathogen Porphyromonas gingivalis and a less pronounced effect for P11-4 hydrogels is demonstrated. The metabolic activity rates of human dental follicle stem cells (DFSCs), which reflect cell viability and may thus indicate the regenerative capacity, are similar on tissue culture polystyrene (TCPS) and on P11-4 hydrogels after 14 days of culture. Noticeably, both SAP hydrogels strengthen the osteogenic differentiation of DFSCs compared with TCPS. The incorporation of tetracycline, ciprofloxacin, and doxycycline does not affect fibril formation of either SAP hydrogel and results in favorable release kinetics up to 120 h. In summary, this study reveals that P11-SAP hydrogels combine many favorable properties required to make them applicable as prospective novel treatment strategy for periodontal therapy.

Comparing Physical Container Closure Integrity Test Methods and Artificial Leak Methodologies.

The sterility of drug products intended for parenteral administration is a critical quality attribute (CQA) because it serves to ensure patient safety and is thus a key requirement by health authorities. While sterility testing is a probabilistic test, the assurance of sterility is a holistic concept including adequate design of manufacturing facilities, process performance, and product design. Container closure integrity testing (CCIT) is necessary to confirm the integrity of a container closure system (CCS), until the end of a product's shelf life. The new and revised United States Pharmacopeia (USP) General Chapter <1207> is a comprehensive guidance on CCI. Nevertheless, practical considerations including the choice of CCIT methods, the acceptance criteria, or the positive control samples (artificial leaks) must be addressed by the pharmaceutical manufacturer.This study is the first to provide a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA), and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).The results from these experiments provide comprehensive data to allow a direct comparison of the capabilities of the individual methods. The results confirmed that the He leak detection method, which is considered the "gold-standard" for pCCIT regarding method sensitivity, indeed demonstrates the highest detection sensitivity (lowest detection limit). In comparison to the dye ingress method, HSA and vacuum decay also demonstrated better detection sensitivity in our study.Capillary leaks with orifice diameter (capillary leak with flow according to an ideal orifice) and micro holes yielded similar leak rates, whereas capillaries with nominal diameters yielded significantly lower leak rates. In conclusion, method sensitivity cannot be compared by means of a leak diameter, but requires the consideration of multiple impacting factors (e.g., path length, uniformity).LAY ABSTRACT: Sterility of drug products intended for parenteral administration is a critical quality attribute to ensure patient's safety and is thus a key requirement by health authorities. The absence of microbial contamination must be demonstrated by container closure integrity (CCI) of the container closure system (CCS). Currently, the revised United States Pharmacopeia (USP) General Chapter <1207> provides the most extensive guidance on how CCI should be assessed. Nevertheless, practical considerations on the choice of an appropriate CCIT method, artificial leaks or the choice of an acceptance criteria are lacking and must be addressed by the pharmaceutical manufacturer.This study provides a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA) and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).

Matrix metalloprotease triggered bioresponsive drug delivery systems - Design, synthesis and application.

Engineering of drug delivery systems has evolved in recent decades from comparably simple designs that merely controlled drug release to complex, often multistage systems that respond to multiple biological or environmental stimuli. Matrix metalloproteases (MMPs) are a family of proteolytic enzymes that are involved in numerous physiologic and pathophysiologic processes, including cancer. Therefore, these enzymes represent highly relevant targets for the development of novel bioresponsive drug delivery systems. The first part of this review summarizes major developments of the various types of MMP responsive drug delivery systems that have been achieved in the last decade and highlights promising strategies. The selection and incorporation of MMP sensitive elements into drug delivery systems as well as the interaction between MMP, drug delivery system and drug require additional scrutiny to avoid common pitfalls. Thus, the second part of this review focusses on strategies for successful selection and incorporation of MMP sensitive elements and on important design parameters related to the drug delivery system and the drug. This review will therefore provide a broad overview of successful MMP-sensitive drug delivery system designs and will inform about important design criteria for novel systems.

Methods To Determine the Silicone Oil Layer Thickness in Sprayed-On Siliconized Syringes.

The silicone lubricant layer in prefilled syringes has been investigated with regards to siliconization process performance, prefilled syringe functionality, and drug product attributes, such as subvisible particle levels, in several studies in the past. However, adequate methods to characterize the silicone oil layer thickness and distribution are limited, and systematic evaluation is missing. In this study, white light interferometry was evaluated to close this gap in method understanding. White light interferometry demonstrated a good accuracy of 93-99% for MgF2 coated, curved standards covering a thickness range of 115-473 nm. Thickness measurements for sprayed-on siliconized prefilled syringes with different representative silicone oil distribution patterns (homogeneous, pronounced siliconization at flange or needle side, respectively) showed high instrument (0.5%) and analyst precision (4.1%). Different white light interferometry instrument parameters (autofocus, protective shield, syringe barrel dimensions input, type of non-siliconized syringe used as base reference) had no significant impact on the measured average layer thickness. The obtained values from white light interferometry applying a fully developed method (12 radial lines, 50 mm measurement distance, 50 measurements points) were in agreement with orthogonal results from combined white and laser interferometry and 3D-laser scanning microscopy. The investigated syringe batches (lot A and B) exhibited comparable longitudinal silicone oil layer thicknesses ranging from 170-190 nm to 90-100 nm from flange to tip and homogeneously distributed silicone layers over the syringe barrel circumference (110- 135 nm). Empty break-loose (4-4.5 N) and gliding forces (2-2.5 N) were comparably low for both analyzed syringe lots. A silicone oil layer thickness of 100-200 nm was thus sufficient for adequate functionality in this particular study. Filling the syringe with a surrogate solution including short-term exposure and emptying did not significantly influence the silicone oil layer at the investigated silicone level. It thus appears reasonable to use this approach to characterize silicone oil layers in filled syringes over time. The developed method characterizes non-destructively the layer thickness and distribution of silicone oil in empty syringes and provides fast access to reliable results. The gained information can be further used to support optimization of siliconization processes and increase the understanding of syringe functionality.LAY ABSTRACT: Silicone oil layers as lubricant are required to ensure functionality of prefilled syringes. Methods evaluating these layers are limited, and systematic evaluation is missing. The aim of this study was to develop and assess white light interferometry as an analytical method to characterize sprayed-on silicone oil layers in 1 mL prefilled syringes. White light interferometry showed a good accuracy (93-99%) as well as instrument and analyst precision (0.5% and 4.1%, respectively). Different applied instrument parameters had no significant impact on the measured layer thickness. The obtained values from white light interferometry applying a fully developed method concurred with orthogonal results from 3D-laser scanning microscopy and combined white light and laser interferometry. The average layer thicknesses in two investigated syringe lots gradually decreased from 170-190 nm at the flange to 100-90 nm at the needle side. The silicone layers were homogeneously distributed over the syringe barrel circumference (110-135 nm) for both lots. Empty break-loose (4-4.5 N) and gliding forces (2-2.5 N) were comparably low for both analyzed syringe lots. Syringe filling with a surrogate solution, including short-term exposure and emptying, did not significantly affect the silicone oil layer. The developed, non-destructive method provided reliable results to characterize the silicone oil layer thickness and distribution in empty siliconized syringes. This information can be further used to support optimization of siliconization processes and increase understanding of syringe functionality.

Container Closure Integrity Testing of Prefilled Syringes.

Prefilled syringes (PFSs) are increasingly preferred over vials as container closure systems (CCSs) for injectable drug products when facilitated or self-administration is required. However, PFSs are more complex compared to CCSs consisting of vial, rubber stopper, and crimp cap. Container closure integrity (CCI) assurance and verification has been a specific challenge for PFSs as they feature several sealing areas. A comprehensive understanding of the CCS is necessary for an appropriate CCI assessment as well as for packaging development and qualification. A comprehensive CCI assessment of 6 different PFSs from 3 different manufacturers (including 1 polymeric PFS) was conducted using helium leak testing. PFS components were manipulated to systematically assess the contribution of the different sealing areas to CCI, namely rigid needle shield (RNS)/needle, RNS/tip cone, and the individual ribs of a syringe plunger. The polymeric PFS required an equilibrium measurement for accurate container closure integrity testing. The different sealing areas and a single plunger rib were shown to provide adequate CCI. Acceptable tip cap movement until the point of CCI failure was estimated. The assessment of acceptable tip cap movement demonstrated the importance of considering the RNS/tip cone seal design to ensure CCI of the PFS upon post assembly possesses and shipment.

Surface modification of nanofibrous matrices via layer-by-layer functionalized silk assembly for mitigating the foreign body reaction.

The inherent hydrophobicity and large surface area of electrospun synthetic polymeric scaffolds often cause non-specific protein adsorption, thereby influencing macrophage functions and eventually leading to fibrosis at the tissue-scaffold interface. This work reports fabrication of silk fibroin (SF)-functionalized electrospun polycaprolactone (PCL) fibers by single-component layer-by-layer assembly and decorate the SF with heparin disaccharide (HD), resulting in the non-covalent binding of interleukin-4 (IL-4) with the capacity to modulate macrophage polarization. A modified SF derivative was obtained by diazonium coupling and then covalently bonded with HD via click chemistry to eventually bind IL-4 efficiently and maintain its bioactivity. In vitro studies showed that IL-4 surface-functionalized nanofibrous scaffolds promoted polarization to M2 macrophages in the short-term. Importantly, in a murine subcutaneous implantation model, we found that promoting transient shifts in macrophage polarization at early stage can significantly inhibit the extent of the late foreign body reactions. Furthermore, the results of a transcriptomic profiling showed that MARK, PI3K, JNK and NF-κB signaling pathways played an important role in regulating the macrophage phenotypes in the SF/HD/IL-4-functionalized fibers. Our results suggest that such a strategy offers a new approach for utilizing biological agent surface functionalization to modulate the foreign body reaction to nanofibrous scaffolds.

Effect of WOW process parameters on morphology and burst release of FITC-dextran loaded PLGA microspheres.

Using fluorescein isothiocyanate labeled dextran (FITC-dextran 40, FD40) as a hydrophilic model compound, microspheres were prepared by a WOW double emulsion technique. Influence of process parameters on microsphere morphology and burst release of FD40 from PLGA microspheres was studied. Internal morphology of microspheres was investigated by stereological method via cryo-cutting technique and scanning electron microscopy (SEM). Drug distribution in microspheres was observed with confocal laser scanning microscopy (CLSM). Polymer nature (RG503 and RG503H) had significant influence on the micro-morphology of microspheres. Increase in continuous water phase volume (W2) led to increased surface porosity but decreased internal porosity. By increasing PVA concentration in the continuous phase from 0.1 to 1%, particle size changed marginally but burst release decreased from 12.2 to 5.9%. Internal porosity of microspheres decreased considerably with increasing polymer concentration. Increase in homogenization speed during the primary emulsion preparation led to decreased internal porosity. Burst release decreased with increasing drug loading but increased with drug molecular weight. Drug distribution in microspheres depended on preparation method. The porosity of microspheres decreased with time in the diffusion stage, but internal morphology had no influence on the release behavior in the bioerosion stage. In summary, surface porosity and internal morphology play a significant role in the release of hydrophilic macromolecules from biodegradable microspheres in the initial release phase characterized by pore diffusion.

Silk fibroin degumming affects scaffold structure and release of macromolecular drugs.

Silk fibroin (SF) is a natural polymer with tremendous potential as a matrix for drug delivery systems as well as for tissue engineering. Silk sericin (SS) removal (degumming) is a critical step during SF purification, potentially affecting SF integrity and resulting in structural changes such as partial hydrolysis and inhibition of micelle formation. In addition to SF composition itself, the molecular weight and charge of encapsulated drugs may significantly affect drug release from SF matrices. The effect of these parameters on drug release was investigated by varying SF degumming time and charge of the model compound encapsulated in SF films. With increasing degumming time, average SF molecular weight decreased, molecular weight distribution became broader and formation of SF micelles was impaired. However, ß-sheet content was not affected by degumming time, suggesting that degradation occurred mainly in hydrophilic domains of SF. The release of differently charged dextran derivatives, used as macromolecular model drugs, was significantly affected by SF degumming. Release of neutral dextran increased with increasing degumming time. In contrast, negatively charged dextran showed an inverse effect potentially due to reduced SF charge density with increased degumming time. Interestingly, positively charged dextran were shown to partly form polyelectrolyte complexes with SF by isothermal titration calorimetry but also exhibited phase separation during film drying resulting in fast burst release. These results demonstrate that both, SF preparation as well as drug charge significantly affect drug release from SF matrices.

Surface functionalization allowing repetitive use of optical sensors for real-time detection of antibody-bacteria interaction.

In this study, sensor surface functionalization allowing the repetitive use of a sensing device was evaluated for antibody-based detection of living bacteria using an optical planar Bragg grating sensor. To achieve regenerable immobilization of bacteria specific antibodies, the heterobifunctional cross-linker N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was linked to an aminosilanized sensor surface and subsequently reduced to expose sulfhydryl groups enabling the covalent conjugation of SPDP-activated antibodies via disulfide bonds. The immobilization of a capture antibody specific for Staphylococcus aureus on the sensor surface as well as specific binding of S. aureus could be monitored, highlighting the applicability of optical sensors for the specific detection of large biological structures. Reusability of bacteria saturated sensors was successfully demonstrated by cleaving the antibody along with bound bacteria through reduction of disulfide bonds and subsequent re-functionalization with activated antibody, resulting in comparable sensitivity towards S. aureus.

Simple and rapid high performance liquid chromatography method for the determination of polidocanol as bulk product and in pharmaceutical polymer matrices using charged aerosol detection.

Currently, neither the European nor the United States Pharmacopoeia provide a method for the determination of polidocanol (PD) content despite the fact that PD, besides being an excipient, is also used as an active pharmaceutical ingredient. We therefore developed a method where the PD content was determined using a Kinetex C18 column operated at 40°C with water-acetonitrile (15:85, v/v) as mobile phase. A Corona(®) charged aerosol detector was employed for the detection of PD that is lacking a suitable UV chromophore. The method was fully validated. Additionally, the method was applied for the determination of PD release from a pharmaceutical polymer matrix consisting of poly-ɛ-caprolactone and poly(lactic-co-glycolic acid) and PD.

Fatty acid composition analysis in polysorbate 80 with high performance liquid chromatography coupled to charged aerosol detection.

The fatty acid (FA) composition of polysorbate 80 (PS80), a sorbitan oleic acid ester copolymerized with about 20mole of ethylene oxide, is typically characterized by gas chromatography. Here, an alternative method was developed. After saponification with potassium hydroxide the FA fraction was collected with liquid-liquid extraction using methyl-tert-butyl ether. HPLC in combination with a Corona® charged aerosol detector (CAD) was applied for the separation and detection. The method was fully validated in terms of specificity, repeatability, limits of quantification, linearity, range, accuracy and robustness. The characterization of 16 different PS80 batches demonstrated variability regarding their FA composition, with e.g. the amount of oleic acid ranging from 67.8±0.7% to 96.6±1.4%. Furthermore, we identified petroselinic acid, a double-bond positional isomer to oleic acid in all batches, an FA not known to pharmacopoeias at present. In addition, 11-hydroxy-9-octadecenoic acid, an oxidation product of oleic acid was identified. Structure elucidation was performed by means of HPLC-MS/MS. In addition, the method was expanded to the evaluation of the free FAs. Having determined the entire FA composition, the acid value according to EP and USP can be calculated.

Predicting critical micelle concentration and micelle molecular weight of polysorbate 80 using compendial methods.

This manuscript addresses the capability of compendial methods in controlling polysorbate 80 (PS80) functionality. Based on the analysis of sixteen batches, functionality related characteristics (FRC) including critical micelle concentration (CMC), cloud point, hydrophilic-lipophilic balance (HLB) value and micelle molecular weight were correlated to chemical composition including fatty acids before and after hydrolysis, content of non-esterified polyethylene glycols and sorbitan polyethoxylates, sorbitan- and isosorbide polyethoxylate fatty acid mono- and diesters, polyoxyethylene diesters, and peroxide values. Batches from some suppliers had a high variability in functionality related characteristic (FRC), questioning the ability of the current monograph in controlling these. Interestingly, the combined use of the input parameters oleic acid content and peroxide value - both of which being monographed methods - resulted in a model adequately predicting CMC. Confining the batches to those complying with specifications for peroxide value proved oleic acid content alone as being predictive for CMC. Similarly, a four parameter model based on chemical analyses alone was instrumental in predicting the molecular weight of PS80 micelles. Improved models based on analytical outcome from fingerprint analyses are also presented. A road map controlling PS80 batches with respect to FRC and based on chemical analyses alone is provided for the formulator.

The effect of silk gland sericin protein incorporation into electrospun polycaprolactone nanofibers on in vitro and in vivo characteristics.

The application of silk fibroin is a promising approach for designing biomaterials. However, silk sericin (SS) protein has not attracted much attention in the field of biomaterials as a natural biopolymer due to its immune responses, weak structural properties and high solubility. In this study, fifth instar silkworm (B. mori) middle gland extracted sericin protein and polycaprolactone (PCL) blends nanofibrous scaffolds were successfully fabricated via an emulsion electrospinning technique. PCL/SS nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Water contact angle and tensile measurements indicated that the PCL/SS scaffolds exhibited improved mechanical properties, as well as more favorable wettability, than that obtained from PCL alone. We also analyzed the effect of SS content in blends on cell morphology and proliferation of human primary skin fibroblasts (FEK4 cells) within 1-5 days. The results showed that cell proliferation significantly increased in the appropriate ratio of PCL/SS blends while showing more elongated cellular morphology. The mRNA gene expression of transforming growth factor ß1 (TGF-ß1) and collagen I were up-regulated in PCL/SS scaffolds. Furthermore, in vivo experiments suggested that low fibrosis tissue formation and macrophages adhesion of the PCL/SS nanofibrous scaffolds reveal its potential as future biocompatible scaffolds for tissue engineering.