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.

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.

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.

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.

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.

Neutron activation of holmium poly(L-lactic acid) microspheres for hepatic arterial radio-embolization: a validation study.

Poly(L-lactic acid) microspheres loaded with holmium-166 acetylacetonate (166Ho-PLLA-MS) are a novel microdevice for intra-arterial radio-embolization in patients with unresectable liver malignancies. The neutron activation in a nuclear reactor, in particular the gamma heating, damages the 166Ho-PLLA-MS. The degree of damage is dependent on the irradiation characteristics and irradiation time in a particular reactor facility. The aim of this study was to standardize and objectively validate the activation procedure in a particular reactor. The methods included light- and scanning electron microscopy (SEM), particle size analysis, differential scanning calorimetry, viscometry, thermal neutron flux measurements and energy deposition calculations. Seven hours-neutron irradiation results in sufficient specific activity of the 166Ho-PLLA-MS while structural integrity is preserved. Neutron flux measurements and energy deposition calculations are required in the screening of other nuclear reactors. For the evaluation of microsphere quality, light microscopy, SEM and particle size analysis are appropriate techniques.

Microspheres with ultrahigh holmium content for radioablation of malignancies.

PURPOSE: The aim of this study was to develop microspheres with an ultra high holmium content which can be neutron activated for radioablation of malignancies. These microspheres are proposed to be delivered selectively through either intratumoral injections into solid tumors or administered via an intravascularly placed catheter. METHODS: Microspheres were prepared by solvent evaporation, using holmium acetylacetonate (HoAcAc) crystals as the sole ingredient. Microspheres were characterized using light and scanning electron microscopy, coulter counter, titrimetry, infrared and Raman spectroscopy, differential scanning calorimetry, X-ray powder diffraction, magnetic resonance imaging (MRI), and X-ray computed tomography (CT). RESULTS: Microspheres, thus prepared displayed a smooth surface. The holmium content of the HoAcAc microspheres (44% (w/w)) was higher than the holmium content of the starting material, HoAcAc crystals (33% (w/w)). This was attributed to the loss of acetylacetonate from the HoAcAc complex, during rearrangement of acetylacetonate around the holmium ion. The increase of the holmium content allows for the detection of (sub)microgram amounts of microspheres using MRI and CT. CONCLUSIONS: HoAcAc microspheres with an ultra-high holmium content were prepared. These microspheres are suitable for radioablation of tumors by intratumoral injections or treatment of liver tumors through transcatheter administration.

Tumour-targeted nanomedicines: principles and practice.

Drug targeting systems are nanometre-sized carrier materials designed for improving the biodistribution of systemically applied (chemo)therapeutics. Various different tumour-targeted nanomedicines have been evaluated over the years, and clear evidence is currently available for substantial improvement of the therapeutic index of anticancer agents. Here, we briefly summarise the most important targeting systems and strategies, and discuss recent advances and future directions in the development of tumour-targeted nanomedicines.

Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression.

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.

Renal targeting of kinase inhibitors.

Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.