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Graphical abstract Abstract 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β1 (TGF-β1) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sericin removal from silk (degumming) affects material characteristics of silk fibroin (SF). Sodium carbonate is most commonly used for degumming, but numerous alternative methods are available. Herein, a systematic comparison of degumming methods is provided. Sodium carbonate, sodium oleate, trypsin, and ionic liquid are used, and materials are characterized regarding mass loss, SF content, molecular integrity of SF, refractive index, and tensile properties. Complete degumming is achieved within 30 min of using sodium carbonate, but results in significant reduction of molecular weight, shift toward less acidic charge variants, and reduction of yield‐ and rupture force. Sodium oleate and trypsin are inefficient and negatively affect tensile properties, while ionic liquid shows good efficiency and marginal degradation of SF but also reduced yield‐ and rupture force. Refractive index is not affected by degumming. These results allow rational selection of the degumming method and tuning of SF properties for biomedical applications. The degumming of virgin silk significantly influences the physicochemical properties and material characteristics of resulting silk fibroin. The efficiency of different degumming methods and their effect on mass loss, silk fibroin content, molecular weight, and charge as well as on tensile and optical properties is investigated to allow rational process selection for biomedical applications. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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)., (© PDA, Inc. 2019.)

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 MgF 2 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., (© PDA, Inc. 2018.)

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.

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.

Knowledge of the influence of targeting ligands on pharmacokinetics and biodistribution of polymeric nonviral vectors is presently limited. We investigated the properties of three structurally different conjugates of polyethylenglycol-modified polyethylenimine coupled to the HER2 specific antibody Trastuzumab. Unlike polyethylenimine, conjugates formed small (100-230 nm) DNA polyplexes with zeta-potentials of +/- 2 mV at a broad range of N/P ratios. Stability as assessed by heparin displacement was slightly improved compared to unmodified copolymers. Erythrocyte aggregation and hemolysis were strongly reduced with conjugates. Conjugate polyplexes showed significant differences in specificity and transfection efficiency in vitro. These could be attributed to differences in cell binding and uptake assessed by flow cytometry. Pharmacokinetics of conjugates in mice revealed significant improvements over free plasmid DNA and polyethylenimine. Area under the plasma level-time curve of conjugates was increased up to 48% or 114% compared to that of polyethylenimine or free plasmid DNA, respectively. Deposition of conjugate polyplexes in lung and spleen was significantly reduced compared to that of polyethylenimine. Differences could be attributed to antibody conjugation since no significant differences in pharmacokinetics and biodistribution were found between conjugates. These findings demonstrate that conjugated antibodies not only confer active targeting but also significantly improve in vivo properties of polyplexes.

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.

In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2,000 Da)(10)-graft-poly(ethylene imine 25 kDa) (PEG-PEI) covalently coupled to Trastuzumab (Herceptin) via N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) for specific gene delivery to Her2-expressing cell lines. The efficiency of DNA condensation was studied using an ethidium bromide exclusion assay and demonstrated negligible differences compared to PEG-PEI. Conjugate complex sizes were determined by dynamic light scattering to be in the range 130-180 nm. zeta potentials at different N/P ratios were close to neutral. Flow cytometry and confocal microscopy revealed efficient binding and uptake of Trastuzumab-PEI-PEG complexes using Her2-positive SK-BR-3 cells. In contrast, binding and uptake into Her2-negative OVCAR-3 cells was negligible. In good correlation with these findings, reporter gene expression using targeted complexes in SK-BR-3 cells was up to sevenfold higher than that of unmodified PEG-PEI complexes. With the use OVCAR-3 cells, no significant difference in expression efficiencies could be observed between conjugate and PEG-PEI complexes. Inhibition experiments with free Trastuzumab showed a significant decrease in reporter gene expression using SK-BR-3 cells but no decrease using OVCAR-3 cells, strongly supporting a specific Her2-receptor-mediated uptake mechanism. Our results suggest that Trastuzumab-PEI-PEG might be a promising new bioconjugate for targeted gene transfer to Her2-positive tumor cells in vivo.