Development of a compounded propofol nanoemulsion using multiple non-invasive process analytical technologies.

Propofol is the preferred anaesthetic for induction and maintenance of sedation in critically ill mechanically ventilated COVID-19 patients. However, during the outbreak of the COVID-19 pandemic, regular supply chains could not keep up with the sudden increase in global demand, causing drug shortages. Propofol is formulated as an oil-in-water emulsion which is administered intravenously. This study explores the extemporaneous preparation of a propofol emulsion without specialized manufacturing equipment to temporally alleviate such shortages. A commercially available lipid emulsion (IVLE, SMOFlipid 20 %), intended for parenteral nutrition, was used to create a propofol loaded nanoemulsion via addition of liquid propofol drug substance and subsequent mixing. Critical quality attributes such as mean droplet size and the volume-weighted percentage of large-diameter (>5µm) droplets were studied. The evolution of droplet size and propofol distribution was monitored in situ and non-destructively, maintaining sterility, using Spatially Resolved Dynamic Light Scattering and Near Infrared Spectroscopy, respectively. Using response surface methodology, an optimum was found for a 4 % w/v propofol formulation with a ∼15 min mixing time in a flask shaker at a 40° shaking angle. This study shows that extemporaneous compounding is a viable option for emergency supply of propofol drug product during global drug shortages.

Preparation and characterization of inorganic radioactive holmium-166 microspheres for internal radionuclide therapy.

Microspheres with high specific activities of radionuclides are very interesting for internal radiotherapy treatments. This work focuses on the formulation and characterization of inorganic microspheres with a high content of holmium and therefore a high specific radioactivity of holmium-166. Two novel formulations of inorganic microspheres were obtained by dispersing solid holmium acetylacetonate microspheres (Ho2(AcAc)3-ms) in NaH2PO4 or NaOH solutions followed by 2 h incubation at room temperature. By exchange of acetylacetonate with phosphate or hydroxyl ions, holmium phosphate microspheres (HoPO4-ms) and holmium hydroxide microspheres (Ho(OH)3-ms) were formed respectively. The inorganic microspheres had a significantly smaller diameter (28.5 ±â€¯4.4 µm (HoPO4-ms) and 25.1 ±â€¯3.5 µm (Ho(OH)3-ms)) than those of Ho2(AcAc)3-ms (32.6 ±â€¯5.2 µm). The weight percentage of holmium-165 in the microspheres increased significantly from 47% (Ho2(AcAc)3-ms) to 55% (HoPO4-ms) and 73% (Ho(OH)3-ms). After preparation of both HoPO4-ms and Ho(OH)3-ms, the stable holmium-165 isotope was partly converted by neutron activation into radioactive holmium-166 to yield radioactive microspheres. High specific activities were achieved ranging from 21.7 to 59.9 MBq/mg (166HoPO4-ms) and from 28.8 to 79.9 MBq/mg (166Ho(OH)3-ms) depending on the neutron activation time. The structure of both microspheres was preserved up to neutron activations of 6 h in a thermal neutron flux of 4.72 × 1016 n m-2 s-1. After activation, both microspheres revealed excellent stability in administration fluids (saline and phosphate buffer) having less than 0.05% of holmium released after 72 h incubation. Finally, the hemocompatibility of these inorganic microspheres was evaluated and it was shown that the microspheres did cause neither hemolysis nor depletion or inhibition of the coagulation factors of the intrinsic blood coagulation pathway meaning that the microspheres have a good hemocompatibility. Overall, this work shows that radioactive inorganic microspheres with high specific activities of holmium-166 can be prepared which potentially can be used for internal radionuclide therapy.

Radioactive holmium phosphate microspheres for cancer treatment.

The aim of this study was the development of radioactive holmium phosphate microspheres (HoPO4-MS) with a high holmium content and that are stable in human serum for selective internal radiation therapy (SIRT) of liver cancer. To this end, holmium acetylacetonate microspheres (HoAcAc-MS) were prepared (34.2 ±â€¯1.0 µm in diameter, holmium content of 46.2 ±â€¯0.8 and density of 1.7 g/cm3) via an emulsification and solvent evaporation method. The concentration of HoAcAc in the organic solvent, the temperature of emulsification and the stirring speed were varied for the preparation of the HoAcAc-MS to obtain microspheres with different diameters ranging from 11 to 35 µm. Subsequently, the AcAc ligands of the HoAcAc-MS were replaced by phosphate ions by simply incubating neutron irradiated HoAcAc-MS in a phosphate buffer solution (0.116 M, pH 4.2) to yield radioactive HoPO4-MS. The obtained microspheres were analyzed using different techniques such as SEM-EDS, ICP-OES and HPLC. The prepared HoPO4-MS (29.5 ±â€¯1.2 µm in diameter and a density of 3.1 g/cm3) present an even higher holmium content (52 wt%) than the HoAcAc-MS precursor (46 wt%). Finally, the stability of the HoPO4-MS was tested by incubation in human serum at 37 °C which showed no visible changes of the microspheres morphology and only 0.1% of holmium release was observed during the 2 weeks period of incubation. In conclusion, this study shows that stable radioactive HoPO4-MS can be prepared with suitable properties to be used for cancer therapy.

Assessing bioink shape fidelity to aid material development in 3D bioprinting.

During extrusion-based bioprinting, the deposited bioink filaments are subjected to deformations, such as collapse of overhanging filaments, which compromises the ability to stack several layers of bioink, and fusion between adjacent filaments, which compromises the resolution and maintenance of a desired pore structure. When developing new bioinks, approaches to assess their shape fidelity after printing would be beneficial to evaluate the degree of deformation of the deposited filament and to estimate how similar the final printed construct would be to the design. However, shape fidelity has been prevalently assessed qualitatively through visual inspection after printing, hampering the direct comparison of the printability of different bioinks. In this technical note, we propose a quantitative evaluation for shape fidelity of bioinks based on testing the filament collapse on overhanging structures and the filament fusion of parallel printed strands. Both tests were applied on a hydrogel platform based on poloxamer 407 and poly(ethylene glycol) blends, providing a library of hydrogels with different yield stresses. The presented approach is an easy way to assess bioink shape fidelity, applicable to any filament-based bioprinting system and able to quantitatively evaluate this aspect of printability, based on the degree of deformation of the printed filament. In addition, we built a simple theoretical model that relates filament collapse with bioink yield stress. The results of both shape fidelity tests underline the role of yield stress as one of the parameters influencing the printability of a bioink. The presented quantitative evaluation will allow for reproducible comparisons between different bioink platforms.

Overcoming multidrug resistance using folate receptor-targeted and pH-responsive polymeric nanogels containing covalently entrapped doxorubicin.

Multidrug resistance (MDR) contributes to failure of chemotherapy. We here show that biodegradable polymeric nanogels are able to overcome MDR via folic acid targeting. The nanogels are based on hydroxyethyl methacrylamide-oligoglycolates-derivatized poly(hydroxyethyl methacrylamide-co-N-(2-azidoethyl)methacrylamide) (p(HEMAm-co-AzEMAm)-Gly-HEMAm), covalently loaded with the chemotherapeutic drug doxorubicin (DOX) and subsequently decorated with a folic acid-PEG conjugate via copper-free click chemistry. pH-Responsive drug release is achieved via the acid-labile hydrazone bond between DOX and the methacrylamide polymeric network. Cellular uptake and cytotoxicity analyses in folate receptor-positive B16F10 melanoma versus folate receptor-negative A549 lung carcinoma cells confirmed specific uptake of the targeted nanogels. Confocal microscopy demonstrated efficient internalization, lysosomal trafficking, drug release and nuclear localization of DOX. We also show that DOX resistance in 4T1 breast cancer cells results in upregulation of the folate receptor, and that folic acid targeted nanogels can be employed to bypass drug efflux pumps, resulting in highly efficient killing of resistant cancer cells. In conclusion, folic acid functionalized nanogels with pH-controlled drug release seem to hold significant potential for treating multidrug resistant malignancies.

Simultaneous Delivery of Multiple Antibacterial Agents from Additively Manufactured Porous Biomaterials to Fully Eradicate Planktonic and Adherent Staphylococcus aureus.

Implant-associated infections are notoriously difficult to treat and may even result in amputation and death. The first few days after surgery are the most critical time to prevent those infections, preferably through full eradication of the micro-organisms entering the body perioperatively. That is particularly important for patients with a compromised immune system such as orthopedic oncology patients, as they are at higher risk for infection and complications. Full eradication of bacteria is, especially in a biofilm, extremely challenging due to the toxicity barrier that prevents delivery of high doses of antibacterial agents. This study aimed to use the potential synergistic effects of multiple antibacterial agents to prevent the use of toxic levels of these agents and achieve full eradication of planktonic and adherent bacteria. Silver ions and vancomycin were therefore simultaneously delivered from additively manufactured highly porous titanium implants with an extremely high surface area incorporating a bactericidal coating made from chitosan and gelatin applied by electrophoretic deposition (EPD). The presence of the chitosan/gelatin (Ch+Gel) coating, Ag, and vancomycin (Vanco) was confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The release of vancomycin and silver ions continued for at least 21 days as measured by inductively coupled plasma (ICP) and UV-spectroscopy. Antibacterial behavior against Staphylococcus aureus, both planktonic and in biofilm, was evaluated for up to 21 days. The Ch+Gel coating showed some bactericidal behavior on its own, while the loaded hydrogels (Ch+Gel+Ag and Ch+Gel+Vanco) achieved full eradication of both planktonic and adherent bacteria without causing significant levels of toxicity. Combining silver and vancomycin improved the release profiles of both agents and revealed a synergistic behavior that further increased the bactericidal effects.

Development of a thermosensitive HAMA-containing bio-ink for the fabrication of composite cartilage repair constructs.

Fine-tuning of bio-ink composition and material processing parameters is crucial for the development of biomechanically relevant cartilage constructs. This study aims to design and develop cartilage constructs with tunable internal architectures and relevant mechanical properties. More specifically, the potential of methacrylated hyaluronic acid (HAMA) added to thermosensitive hydrogels composed of methacrylated poly[N-(2-hydroxypropyl)methacrylamide mono/dilactate] (pHPMA-lac)/polyethylene glycol (PEG) triblock copolymers, to optimize cartilage-like tissue formation by embedded chondrocytes, and enhance printability was explored. Additionally, co-printing with polycaprolactone (PCL) was performed for mechanical reinforcement. Chondrocyte-laden hydrogels composed of pHPMA-lac-PEG and different concentrations of HAMA (0%-1% w/w) were cultured for 28 d in vitro and subsequently evaluated for the presence of cartilage-like matrix. Young's moduli were determined for hydrogels with the different HAMA concentrations. Additionally, hydrogel/PCL constructs with different internal architectures were co-printed and analyzed for their mechanical properties. The results of this study demonstrated a dose-dependent effect of HAMA concentration on cartilage matrix synthesis by chondrocytes. Glycosaminoglycan (GAG) and collagen type II content increased with intermediate HAMA concentrations (0.25%-0.5%) compared to HAMA-free controls, while a relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. Young's moduli of generated hydrogel constructs ranged from 14 to 31 kPa and increased with increasing HAMA concentration. The pHPMA-lac-PEG hydrogels with 0.5% HAMA were found to be optimal for cartilage-like tissue formation. Therefore, this hydrogel system was co-printed with PCL to generate porous or solid constructs with different mesh sizes. Young's moduli of these composite constructs were in the range of native cartilage (3.5-4.6 MPa). Interestingly, the co-printing procedure influenced the mechanical properties of the final constructs. These findings are relevant for future bio-ink development, as they demonstrate the importance of selecting proper HAMA concentrations, as well as appropriate print settings and construct designs for optimal cartilage matrix deposition and final mechanical properties of constructs, respectively.

Hydrogel-based reinforcement of 3D bioprinted constructs.

Progress within the field of biofabrication is hindered by a lack of suitable hydrogel formulations. Here, we present a novel approach based on a hybrid printing technique to create cellularized 3D printed constructs. The hybrid bioprinting strategy combines a reinforcing gel for mechanical support with a bioink to provide a cytocompatible environment. In comparison with thermoplastics such as [Formula: see text]-polycaprolactone, the hydrogel-based reinforcing gel platform enables printing at cell-friendly temperatures, targets the bioprinting of softer tissues and allows for improved control over degradation kinetics. We prepared amphiphilic macromonomers based on poloxamer that form hydrolysable, covalently cross-linked polymer networks. Dissolved at a concentration of 28.6%w/w in water, it functions as reinforcing gel, while a 5%w/w gelatin-methacryloyl based gel is utilized as bioink. This strategy allows for the creation of complex structures, where the bioink provides a cytocompatible environment for encapsulated cells. Cell viability of equine chondrocytes encapsulated within printed constructs remained largely unaffected by the printing process. The versatility of the system is further demonstrated by the ability to tune the stiffness of printed constructs between 138 and 263 kPa, as well as to tailor the degradation kinetics of the reinforcing gel from several weeks up to more than a year.

A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications.

The aim of this study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. CSMA was synthesized by reaction of chondroitin sulfate with glycidyl methacrylate (GMA) in dimethylsulfoxide at 50°C and its degree of methacrylation was tunable up to 48.5%, by changing reaction time and GMA feed. Unlike polymer solutions composed of CSMA alone (20% w/w), mixtures based on 2% w/w of CSMA and 18% of M15P10 showed strain-softening, thermo-sensitive and shear-thinning properties more pronounced than those found for polymer solutions based on M15P10 alone. Additionally, they displayed a yield stress of 19.2±7.0Pa. The 3D printing of this hydrogel resulted in the generation of constructs with tailorable porosity and good handling properties. Finally, embedded chondrogenic cells remained viable and proliferating over a culture period of 6days. The hydrogel described herein represents a promising biomaterial for cartilage 3D printing applications.

PEG stabilized DNA - poly(ferrocenylsilane) polyplexes for gene delivery.

Polycationic poly(ferrocenylsilane)s (PFS) with tunable amounts of PEG side chains were used for the condensation of DNA into polyplexes of 110 nm in 5.0 mM HEPES. The PFS-PEG/DNA polyplexes showed negligible aggregation, a strongly reduced protein adsorption, transfection activities comparable with linear polyethyleneimine and an excellent cytocompatibility.

Release and pharmacokinetics of near-infrared labeled albumin from monodisperse poly(d,l-lactic-co-hydroxymethyl glycolic acid) microspheres after subcapsular renal injection.

Subcapsular renal injection is a novel administration method for local delivery of therapeutics for the treatment of kidney related diseases. The aim of this study was to investigate the feasibility of polymeric microspheres for sustained release of protein therapeutics in the kidney and study the subsequent redistribution of the released protein. For this purpose, monodisperse poly(d,l-lactic-co-hydroxymethyl glycolic acid) (PLHMGA) microspheres (40 µm in diameter) loaded with near-infrared dye-labeled bovine serum albumin (NIR-BSA) were prepared by a membrane emulsification method. Rats were injected with either free NIR-BSA or with NIR-BSA loaded microspheres (NIR-BSA-ms) and the pharmacokinetics of the released NIR-BSA was studied for 3 weeks by ex vivo imaging of organs and blood. Quantitative release data were obtained from kidney homogenates and possible metabolism of the protein was investigated by SDS-PAGE analysis of the samples. The ex vivo images showed a rapid decrease of the NIR signal within 24h in kidneys injected with free NIR-BSA, while, importantly, the signal of the labeled protein was still visible at day 21 in kidneys injected with NIR-BSA-ms. SDS-PAGE analysis of the kidney homogenates showed that intact NIR-BSA was released from the microspheres. The locally released NIR-BSA drained to the systemic circulation and subsequently accumulated in the liver, where it was degraded and excreted renally. The in vivo release of NIR-BSA was calculated after extracting the protein from the remaining microspheres in kidney homogenates. The in vivo release rate was faster (89 ± 4% of the loading in 2 weeks) compared to the in vitro release of NIR-BSA (38 ± 1% in 2 weeks). In conclusion, PLHMGA microspheres injected under the kidney capsule provide a local depot from which a formulated protein is released over a prolonged time-period.

Formulation and characterization of microspheres loaded with imatinib for sustained delivery.

The aim of this study was the development of imatinib-loaded poly(d,l-lactide-co-glycolide) (PLGA) microspheres with high loading efficiency which can afford continuous release of imatinib over a prolonged period of time. Imatinib mesylate loaded PLGA microspheres with a size of 6-20 µm were prepared by a double emulsion (W1/O/W2) method using dichloromethane as volatile solvent. It was found that the microspheres were spherical with a non-porous surface; imatinib loading efficiency (LE) was highly dependent on the pH of the external water phase (W2). By increasing the pH of W2 phase above the highest pKa of imatinib (pKa 8.1), at which imatinib is mainly uncharged, the LE increased from 10% to 90% (pH 5.0 versus pH 9.0). Conversely, only 4% of its counter ion, mesylate, was retained in the microspheres at the same condition (pH 9.0). Since mesylate is highly water soluble, it is unlikely that it partitions into the organic phase. We demonstrated, using differential scanning calorimetry (DSC), that imatinib was molecularly dispersed in the polymeric matrix at loadings up to 8.0%. At higher drug loading, imatinib partially crystallized in the matrix. Imatinib microspheres released their cargo during three months by a combination of diffusion through the polymer matrix and polymer erosion. In conclusion, we have formulated imatinib microspheres with high LE and LC. Although we started with a double emulsion of imatinib mesylate, the obtained microspheres contained imatinib base which was mainly molecularly dispersed in the polymer matrix. These microspheres release imatinib over a 3-month period which is of interest for local treatment of cancer.

Sunitinib microspheres based on [PDLLA-PEG-PDLLA]-b-PLLA multi-block copolymers for ocular drug delivery.

Sunitinib is a multi-targeted receptor tyrosine kinase (RTK) inhibitor that blocks several angiogenesis related pathways. The aim of this study was to develop sunitinib-loaded polymeric microspheres that can be used as intravitreal formulation for the treatment of ocular diseases. A series of novel multi-block copolymers composed of amorphous blocks of poly-(D,L-lactide) (PDLLA) and polyethylene glycol (PEG) and of semi-crystalline poly-(L-lactide) (PLLA) blocks were synthesized. Sunitinib-loaded microspheres were prepared by a single emulsion method using dichloromethane as volatile solvent and DMSO as co-solvent. SEM images showed that the prepared microspheres (∼ 30 µm) were spherical with a non-porous surface. Sunitinib-loaded microspheres were studied for their degradation and in-vitro release behavior. It was found that increasing the percentage of amorphous soft blocks from 10% to 30% accelerated the degradation of the multi-block copolymers. Sunitinib microspheres released their cargo for a period of at least 210 days by a combination of diffusion and polymer erosion. The initial burst (release in 24h) and release rate could be tailored by controlling the PEG-content of the multi-block copolymers. Sunitinib-loaded microspheres suppressed angiogenesis in a chicken chorioallantoic membrane (CAM) assay. These microspheres therefore hold promise for long-term suppression of ocular neovascularization.

Biocompatibility of poly(D,L-lactic-co-hydroxymethyl glycolic acid) microspheres after subcutaneous and subcapsular renal injection.

Poly(D,L-lactic-co-hydroxymethyl glycolic acid) (PLHMGA) is a biodegradable copolymer with potential as a novel carrier in polymeric drug delivery systems. In this study, the biocompatibility of PLHMGA microspheres (PLHMGA-ms) was investigated both in vitro in three different cell types (PK-84, HK-2 and PTECs) and in vivo at two implantation sites (by subcutaneous and subcapsular renal injection) in rats. Both monodisperse (narrow size distribution) and polydisperse PLHMGA-ms were prepared with volume weight mean diameter of 34 and 17 µm, respectively. Mono and polydisperse PLHMGA-ms showed good cytocompatibility properties upon 72 h incubation with the cells (100 µg microspheres/600 µL/cell line). A mild foreign body reaction was seen shortly after subcutaneous injection (20 mg per pocket) of both mono and polydisperse PLHMGA-ms with the presence of mainly macrophages, few foreign body giant cells and myofibroblasts. This transient inflammatory reaction diminished within 28 days after injection, the time-point at which the microspheres were degraded. The degradation profile is comparable to the in vitro degradation time of the microspheres (i.e., within 35 days) when incubated at 37 °C in phosphate buffered saline. Subcapsular renal injection of monodisperse PLHMGA-ms (10 mg) in rats was characterized with similar inflammatory patterns compared to the subcutaneous injection. No cortical damage was observed in the injected kidneys. In conclusion, this study demonstrates that PLHMGA-ms are well tolerated after in vivo injection in rats. This makes them a good candidate for controlled delivery systems of low-molecular weight drugs as well as protein biopharmaceuticals.

The effect of lauryl capping group on protein release and degradation of poly(D,L-lactic-co-glycolic acid) particles.

The aim of this study was to investigate the effect of a specific and frequently used end group (lauryl alcohol) on the protein release and degradation kinetics of poly(DL-lactic-co-glycolic acid) particles of different sizes. Lauryl-capped PLGA and uncapped PLGA (referred to as PLGA-capped and PLGA-COOH, respectively) particles (0.3, 1 and 20 µm) were prepared by a double emulsion solvent evaporation technique. Bovine serum albumin (BSA) was used as a model protein for release studies. During degradation (PBS buffer, pH7.4 at 37°C), a slower dry mass loss was observed for 0.3 µm particles than for particles of 1 and 20 µm. It was further shown that PLGA-capped particles showed slower mass loss likely due to its more hydrophobic nature. It was found that the ester bond hydrolysis rate was substantially slower for PLGA-capped particles and that the rate increased with particle size. Particles showed enrichment in lactic acid content (and thus a decrease in glycolic acid content) in time, and interestingly PLGA-capped particles showed also an enrichment of the lauryl alcohol content. No difference was observed in degradation kinetics between BSA loaded and blank particles. Independent of size, PLGA-COOH based particles showed, after a small burst, a sustained and nearly complete release of BSA during 60-80 days. On the other hand, particles based on PLGA-capped showed a much slower release and exhibited incomplete release, accompanied by the presence of an insoluble residue remaining even after 180 days. FTIR analysis of this residue showed that it contained both polymer and protein. Considering the polymer enrichment in lauryl alcohol, the incomplete release observed for PLGA-capped is likely attributed to interactions between the protein and the lauryl end group. In conclusion, since PLGA-COOH, in contrast to the capped derivative, shows complete degradation as well as quantitative release of an entrapped protein, this polymer is preferred for the design of protein formulations.

Mechanistic studies on the degradation and protein release characteristics of poly(lactic-co-glycolic-co-hydroxymethylglycolic acid) nanospheres.

The purpose of this study was to gain mechanistic insights into the effect of different formulation parameters on the degradation and release behavior of protein-loaded nanoparticulate carrier systems based on an aliphatic polyester with pendant hydroxyl groups, poly(lactic-co-glycolic-hydroxymethyl glycolic acid) (pLGHMGA). Bovine serum albumin (BSA) was used as a model protein. BSA-loaded pLGHMGA nanospheres of 400-700 nm were prepared using a solvent evaporation method using pLGHMGA of different molecular weights and different compositions. Also, the concentration of pLGHMGA in the organic phase was varied. The nanospheres showed a continuous mass loss accompanied by continuous decrease in number average molecular weight, which indicates that the degradation of the nanospheres is by bulk degradation with a rapid release of water-soluble low molecular weight fragments. On the basis of NMR analysis, it is concluded that intramolecular transesterification precedes extensive hydrolysis of the polymer and degradation of the nanospheres. BSA-loaded freeze-dried nanospheres showed a significant burst release of 40-50% of the BSA loading. In contrast, nonfreeze-dried samples showed a small burst of around 10-20%, indicating that freeze-drying induced pore formation. Nonlyophilized nanospheres prepared from pLGHMGA with 64/18/18 lactic/glycolic/hydroxymethylglycolic acid (L/G/HMG) ratio showed a relatively fast release of BSA for the next 30 days. Nanospheres prepared from a more hydrophobic pLGHMGA (74/13/13, L/G/HMG) showed a two-phase release. Circular dichroism analysis showed that the secondary structure of the released protein was preserved. This study shows a correlation between release behavior and particle erosion rate, which can be modulated by the copolymer composition.

Antitumor efficacy of dexamethasone-loaded core-crosslinked polymeric micelles.

In the current study, core-crosslinked polymeric micelles (DEX-PMs) loaded with three different DEX derivatives designed to display different drug release kinetics, were evaluated for cancer therapy and compared to another effective nanomedicine formulation (long-circulating liposomes encapsulating dexamethasone, LCL-DEX). Pharmacokinetic studies with both radiolabeled dexamethasone and polymer showed that these polymeric systems have long circulating half-lives and may accumulate at the tumor site to a higher extent than liposomes. The in vitro drug release profiles and circulating drug levels in the blood stream show that DEX-PMs with dexamethasone covalently entrapped via a sulfone ester-containing linker (DMSL2) have prolonged circulation time and intermediate drug release kinetics compared to the other polymeric DEX-releasing systems. Furthermore, as the free dexamethasone circulating levels were similar when administered as DMSL2-PM or LCL-DEX, these systems were evaluated simultaneously for antitumor efficacy in B16F10 melanoma bearing mice. The corticosteroid-targeted systems inhibited tumor growth to a similar extent and both increased survival compared to free drug. Recently antitumor efficacy of targeted formulations has been correlated with a systemic effect: a decrease of white blood cell count. In this study all three polymeric systems, liposomes as well as free drug had similar effects on the number of circulating white blood cells, although white blood cell counts recovered faster in the group receiving free drug. In conclusion, corticosteroid-targeting with a polymeric system or a liposomal system translates in similar therapeutic effects. The proven high versatility of the PM with possible optimization and adjustment of the drug release to that required by the therapeutic application, clearly demonstrates the potential of these systems for the treatment of chronic inflammatory diseases including cancer.

Imatinib-ULS-lysozyme: a proximal tubular cell-targeted conjugate of imatinib for the treatment of renal diseases.

The anticancer drug imatinib is an inhibitor of the platelet-derived growth factor receptor (PDGFR) kinases, which are involved in the pathogenesis of fibrotic diseases. In the current study we investigated the delivery of imatinib to the proximal tubular cells of the kidneys and evaluated the potential antifibrotic effects of imatinib in tubulointerstitial fibrosis. Coupling of imatinib to the low molecular weight protein lysozyme via the platinum (II)-based linker ULS yielded a 0.8:1 drug-carrier conjugate that rapidly accumulated in the proximal tubular cells upon intravenous and intraperitoneal administration. The bioavailability of intraperitoneally administered imatinib-ULS-lysozyme was 100%. Renal imatinib levels persisted for up to 3 days after a single injection of imatinib-ULS-lysozyme. Compared with an equal dose imatinib mesylate, imatinib-ULS-lysozyme resulted in a 30- and 15-fold higher renal exposure of imatinib, for intravenous and intraperitoneal administration respectively. Imatinib-ULS-lysozyme could not be detected in the heart, which is the organ at risk for side-effects of prolonged treatment with imatinib. The efficacy of imatinib-ULS-lysozyme in the treatment of tubulointerstitial fibrosis was evaluated in the unilateral ureteral obstruction (UUO) model in mice. Three days UUO resulted in all signs of early fibrosis, i.e. an increased deposition of matrix and production of profibrotic factors. Although a moderately increased activity of PDGFR-ß was observed, the profibrotic phenotype could not be inhibited with imatinib mesylate or with imatinib-ULS-lysozyme. Further evaluation of imatinib mesylate and imatinib-ULS-lysozyme is therefore warranted in an animal model of renal disease in which the activation of PDGFR-ß is more pronounced.

Biodegradable microspheres loaded with an anti-Parkinson prodrug: an in vivo pharmacokinetic study.

During chronic treatment with L-dopa (LD), Parkinsonian patients often experience uncontrolled motor complications due to fluctuations of the plasmatic levels of LD that result in pulsatile dopaminergic stimulation. To overcome these plasmatic fluctuations, a novel prodrug of LD, L-dopa-α-lipoic acid (LD-LA), has been proposed as a tool for achieving continuous dopaminergic stimulation. Due to slower susceptibility toward enzymatic conversion by LD-degrading enzymes (such as catechol-O-methyltransferase and monoamine oxidase), the plasma half-life of this prodrug is longer than that of LD. Moreover, the higher lipophilicity of LD-LA over LD promotes its delivery to the CNS, where the resulting levels of dopamine (DA) are kept high for a longer time than after equimolar administration of LD. To further reduce fluctuations in plasma levels of LD, LD-LA has been entrapped into biodegradable polymeric microspheres to be used as a depot system with the aim to prevent prodrug degradation and to obtain a sustained release of the intact compound. In the present work, a formulation of LD-LA loaded microspheres (characterized for drug loading, size, morphology, thermal properties, and in vitro prodrug release) has been administered subcutaneously to rats, and the resulting levels of LD and DA in plasma and striatal tissue, respectively, have been monitored. A good correlation between the in vitro release kinetics and the time range during which the formulation alters the LD/DA tissue levels in vivo was observed, suggesting that the polymeric microsphere matrix protects the loaded prodrug from chemical and enzymatic degradation and controls its release. Interestingly, LD-LA microspheres provided sustained levels of DA neurotransmitter in the striatum nucleus for up to 4 days after a single administration. In conclusion, a polymeric microsphere formulation of LD-LA is an attractive medicine for treating Parkinson's disease (PD) symptoms, avoiding motor complications.

Synthesis and characterization of biodegradable and thermosensitive polymeric micelles with covalently bound doxorubicin-glucuronide prodrug via click chemistry.

Doxorubicin is an anthracycline anticancer agent that is commonly used in the treatment of a variety of cancers, but its application is associated with severe side effects. Biodegradable and thermosensitive polymeric micelles based on poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAmLac(n))) have been studied as drug delivery systems for therapeutic and imaging agents and have shown promising in vitro and in vivo results. The purpose of this study was to investigate the covalent coupling of a doxorubicin-glucuronide prodrug (DOX-propGA3) to the core of mPEG-b-p(HPMAmLac(2)) micelles. This prodrug is specifically activated by human ß-glucuronidase, an enzyme that is overexpressed in necrotic tumor areas. To this end, an azide modified block copolymer (mPEG(5000)-b-p(HPMAmLac(2)-r-AzEMA)) was synthesized and characterized, and DOX-propGA3 was coupled to the polymer via click chemistry with a high (95%) coupling efficiency. Micelles formed by this DOX containing polymer were small (50 nm) and monodisperse and released 40% of the drug payload after 5 days incubation at 37 °C in the presence of ß-glucuronidase, but less than 5% in the absence of the enzyme. In vitro cytotoxicity experiments demonstrated that DOX micelles incubated with 14C cells showed the same cytotoxicity as free DOX only in the presence of ß-glucuronidase, indicating full conversion of the polymer-bound DOX into the parent drug. Overall, this novel system is very promising for enzymatically responsive anticancer therapy.