Pharmacometric analysis is often used to quantify the differences and similarities between formulation prototypes. In the regulatory framework, it plays a significant role in the evaluation of bioequivalence. While non-compartmental analysis provides an unbiased data evaluation, mechanistic compartmental models such as the physiologically-based nanocarrier biopharmaceutics model promise improved sensitivity and resolution for the underlying causes of inequivalence. In the present investigation, both techniques were applied to two nanomaterial-based formulations for intravenous injection, namely, albumin-stabilized rifabutin nanoparticles and rifabutin-loaded PLGA nanoparticles. The antibiotic rifabutin holds great potential for the treatment of severe and acute infections of patients co-infected with human immunodeficiency virus and tuberculosis. The formulations differ significantly in their formulation and material attributes, resulting in an altered biodistribution pattern as confirmed in a biodistribution study in rats. The albumin-stabilized delivery system further undergoes a dose-dependent change in particle size which leads to a small yet significant change in the in vivo performance. A second analysis was conducted comparing the dose fraction-scaled pharmacokinetic profiles of three dose levels of albumin-stabilized rifabutin nanoparticles. The dose strength affects both the nanomaterial-related absorption and biodistribution of the carrier as well as the drug-related distribution and elimination parameters, increasing the background noise and difficulty of detecting inequivalence. Depending on the pharmacokinetic parameter (e.g., AUC, Cmax, Clobs), the relative (percentage) difference from the average observed using non-compartmental modeling ranged from 85% to 5.2%. A change in the formulation type (PLGA nanoparticles vs. albumin-stabilized rifabutin nanoparticles) resulted in a similar level of inequivalence as compared to a change in the dose strength. A mechanistic compartmental analysis using the physiologically-based nanocarrier biopharmaceutics model led to an average difference of 152.46% between the two formulation prototypes. Albumin-stabilized rifabutin nanoparticles tested at different dose levels led to a 128.30% difference, potentially due to changes in particle size. A comparison of different dose strengths of PLGA nanoparticles, on average, led to a 3.87% difference. This study impressively illustrates the superior sensitivity of mechanistic compartmental analysis when dealing with nanomedicines.

Polylactide-co-glycolide (PLG) nanoparticles hold immense promise for cancer therapy due to their enhanced efficacy and biodegradable matrix structure. Understanding their interactions with blood cells and subsequent biodistribution kinetics is crucial for optimizing their therapeutic potential. In this study, three doxorubicin-loaded PLG nanoparticle systems are synthesized and characterized, analyzing their size, zeta potential, morphology, and in vitro release behavior. Employing intravital microscopy in 4T1-tumor-bearing mice, real-time blood and tumor distribution kinetics are investigated. A mechanistic pharmacokinetic model is used to analyze biodistribution kinetics. Additionally, flow cytometry is utilized to identify cells involved in nanoparticle hitchhiking. Following intravenous injection, PLG nanoparticles exhibit an initial burst release (<1 min) and rapidly adsorb to blood cells (<5 min), hindering extravasation. Agglomeration leads to the clearance of one carrier species within 3 min. In stable dispersions, drug release rather than extravasation remains the dominant pathway for drug elimination from circulation. This comprehensive investigation provides valuable insights into the interplay between competing kinetics that influence the lifecycle of PLG nanoparticles post-injection. The findings advance the understanding of nanoparticle behavior and lay the foundation for improved cancer therapy strategies using nanoparticle-based drug delivery systems.

During the drug development process, many pharmacologically active compounds are discarded because of poor water solubility, but nanoparticle-based formulations are increasingly proposed as a solution for this problem. We therefore studied the distribution of nanoparticulate carriers and the delivery of their poorly water-soluble cargo to a structure of the central nervous system, the retina, under naive and pathological conditions. The lipophilic fluorescent dye coumarin 6 (Cou6) was encapsulated into poly(lactic-co-glycolic acid) PLGA nanoparticles (NPs). After intravenous administration in rats, we analyzed the distribution of cargo Cou6 and of the NP carrier covalently labeled with Cy5.5 in healthy animals and animals with optic nerve crush (ONC). In vivo real-time retina imaging revealed that Cou6 was rapidly released from PLGA NPs and penetrated the inner bloodretina barrier (BRB) within 15 min and PLGA NPs were gradually eliminated from the retinal blood circulation. Ex vivo microscopy of retinal flat mounts indicated that the Cou6 accumulated predominantly in the extracellular space and to a lesser extent in neurons. While the distribution of Cou6 in healthy animals and post ONC was comparable at early time point post-operation, the elimination of the NPs from the vessels was faster on day 7 post ONC. These results demonstrate the importance of considering different kinetics of nano-carrier and poorly water-soluble cargo, emphasizing the critical role of their parenchymal distribution, i.e. cellular/extracellular, and function of different physiological and pathological conditions.

Targeted delivery of doxorubicin still poses a challenge with regards to the quantities reaching the target site as well as the specificity of the uptake. In the present approach, two colloidal nanocarrier systems, NanoCore-6.4 and NanoCore-7.4, loaded with doxorubicin and characterized by different drug release behaviors were evaluated in vitro and in vivo. The nanoparticles utilize a specific surface design to modulate the lipid corona by attracting blood-borne apolipoproteins involved in the endogenous transport of chylomicrons across the blood–brain barrier. When applying this strategy, the fine balance between drug release and carrier accumulation is responsible for targeted delivery. Drug release experiments in an aqueous medium resulted in a difference in drug release of approximately 20%, while a 10% difference was found in human serum. This difference affected the partitioning of doxorubicin in human blood and was reflected by the outcome of the pharmacokinetic study in rats. For the fast-releasing formulation NanoCore-6.4, the AUC0→1h was significantly lower (2999.1 ng × h/mL) than the one of NanoCore-7.4 (3589.5 ng × h/mL). A compartmental analysis using the physiologically-based nanocarrier biopharmaceutics model indicated a significant difference in the release behavior and targeting capability. A fraction of approximately 7.310–7.615% of NanoCore-7.4 was available for drug targeting, while for NanoCore-6.4 only 5.740–6.057% of the injected doxorubicin was accumulated. Although the targeting capabilities indicate bioequivalent behavior, they provide evidence for the quality-by-design approach followed in formulation development.

Fluorescently labeled nanoparticles are widely used for evaluating their distribution in the biological environment. However, dye leakage can lead to misinterpretations of the nanoparticles’ biodistribution. To better understand the interactions of dyes and nanoparticles and their biological environment, we explored PLGA nanoparticles labeled with four widely used dyes encapsulated (coumarin 6, rhodamine 123, DiI) or bound covalently to the polymer (Cy5.5.). The DiI label was stable in both aqueous and lipophilic environments, whereas the quick release of coumarin 6 was observed in model media containing albumin (42%) or liposomes (62%), which could be explained by the different affinity of these dyes to the polymer and lipophilic structures and which we also confirmed by computational modeling (log PDPPC/PLGA: DiI—2.3, Cou6—0.7). The importance of these factors was demonstrated by in vivo neuroimaging (ICON) of the rat retina using double-labeled Cy5.5/Cou6-nanoparticles: encapsulated Cou6 quickly leaked into the tissue, whereas the stably bound Cy.5.5 label remained associated with the vessels. This observation is a good example of the possible misinterpretation of imaging results because the coumarin 6 distribution creates the impression that nanoparticles effectively crossed the blood–retina barrier, whereas in fact no signal from the core material was found beyond the blood vessels.

Introduction. Doxorubicin (Dox) in the composition of poly(lactic-co-glycolic acid, 50:50) (PLGA) – nanoparticles has high antitumor efficacy in rats with glioblastoma 101.8. However, the toxic effect of Dox-PLGA is not well understood. The aim of the study was morphological and biochemical evaluation of the hepatotoxic and cardiotoxic effects of doxorubicin and Dox-PLGA in the glioblastoma 101.8 treat-ment in Wistar rats. Materials and methods. The study was performed on 24 male Wistar rats with glioblastoma: no treatment (n=7), treated with doxorubicin (n=9) or Dox-PLGA (n=8) intravenously at a dose of 1.5 mg/kg on days 2, 5 and 8 after tumor implantation. On the 14th day of the experiment, morphological changes in the myocardium and liver were examined. Hematological and biochemical blood tests were performed. Results.When treating rats with experimental glioblastoma 101.8, Dox-PLGA in comparison with doxorubicin has less pronounced cardiotoxic and hepatotoxic effects according to the morphological, hematological and biochemical tests. Inflammatory changes in the myocardium of the animals treated with Dox-PLGA were less pronounced and widespread than the ones treated with doxorubicin. The activities of total and cardiac creatine phosphokinase (CPK) isoforms and AST were statistically significantly lower in Dox-PLGA group than in animals with glioblastoma without treatment and receiving doxorubicin. The hepatotoxic effects of Dox-PLGA were minimal. Unlike animals treated with doxorubicin, they had mild hepatocyte dystrophy. ALT activity in all groups did not differ from the reference values. Conclusion. Compared with doxorubicin, the nanosomal form of Dox-PLGA in the experimental glioblas-toma 101.8 treatment has less pronounced cardio- and hepatotoxic effects. Keywords: rat glioblastoma 101.8, doxorubicin, PLGA-nanoparticles, toxic effect, liver, myocardium, morphology, biochemistry

The production of polymer nanoparticles with a narrow size distribution on standard T- and X-configuration microfluidic chips with a channel size of 100 μm has been achieved. Introducing ethanol into the aqueous phase in 1:9 ratio (v/v) allowed one to reduce the surface tension of the contacting phases and obtain nanoparticles in the range of 100–150 nm. This makes our approach promising for development of nanoscale drug delivery systems.

The effect of anticancer antibiotic doxorubicin on structural organization of anionic lipid monolayers has been studied. X-ray reflectivity and grazing incidence diffraction techniques were applied to monitor the changes in 2D structure and electron density distribution of Langmuir monolayer composed of negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dioleoylphosphatidylserine (DOPS). For comparison, monolayer of zwitterionic dipalmitoylphosphatidylethanolamine (DPPE) also was investigated. The presented experimental results suggest that doxorubicin interaction with anionic lipid monolayers (DPPG and DOPS) proceeds preferentially via electrostatic attraction—positively charged amino groups of doxorubicin bind to negatively charged head groups of phospholipid molecules. Based on the obtained data, the penetration of doxorubicin into the hydrophobic part of anionic lipid monolayers does not occur. X-ray measurements on DPPE monolayer indicated that doxorubicin did not cause any significant alterations of molecular packing in condensed monolayer of zwitterionic DPPE molecules.

Профиль высвобождения активного ингредиента из наночастиц является одним из важнейших параметров наносомальной лекарственной формы. Разработаны и оптимизированы методы изучения кинетики высвобождения доксорубицина из наночастиц PLGA в модельные среды in vitro. Оптимальным методом отделения наночастиц является ультрацентрифугирование, а наиболее подходящей модельной средой — 1 % водный раствор полоксамера 188. Профиль высвобождения имеет выраженный двухфазный характер и подчиняется закону диффузии Фика. Изучение кинетики высвобождения доксорубицина в плазме крови ограничено низкой стабильностью в условиях эксперимента (4 ч при 37 °C).

Introduction. Drug encapsulation efficiency (EE) is the important parameter of the nanoparticle-based drug formulations. Generally, the methods for evaluation of the EE are based on separation of the free and NP-bound fractions of the drug; however, applicability of these methods for a particulate formulation needs careful consideration.Aim. The purpose of the study was to optimize the procedure for evaluation of the EE for a nanoparticle-based drug formulation taking doxorubicin loaded in the PLGA nanoparticles (PLGA-Dox NP) as a model formulation.Materials and methods. The PLGA-Dox NP were prepared by a “double emulsion” method at pH of the external aqueous phase of 7.4 or 6.4. The NP size and size distribution (PDI) were determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM). For the EE evaluation of doxorubicin, the NP were separated by centrifugation (CF), ultrafiltration (UF), or gel filtration. The efficiency of NP separation by the CF method was evaluated by the PLGA content in the supernatant by capillary electrophoresis (CZE).Results and discussion. The average hydrodynamic diameter of the PLGA-Dox/7.4 and PLGA-Dox/6.4 NP (FCS) was 103 ± 10 nm and 141 ± 8 nm, respectively. According to the TEM data, the main fraction of the NP averaged 50 ± 16 nm. The EE of doxorubicin, determined after the NP separation by centrifugation at 48254.g, was 78.9 ± 1.8 % for the PLGA-Dox/6.4 and 91.5 ± 0.9 % for the PLGA-Dox/7.4 NP with the residual PLGA content in the supernatant of ~5 %. At lower acceleration the NP separation was incomplete leading to underestimation of the EE. Also, the ultrafiltration method using the filters with the NMWL of 50 and 100 kDa enabled the reliable NP separation with the minimal doxorubicin adsorption on the filter (<4 %). Separation of the NP by gel filtration led to the underestimation of the EE due to considerable desorption of doxorubicin from the NP surface.Conclusion. The optimal analytical procedures for evaluation of the EE of doxorubicin in the PLGA NP are based on the NP separation by CF at 48254×g and UF using filters with NMWL of 50 and 100 kDa.

Разработан метод получения водорастворимых форм этопозида солюбилизацией его человеческим сывороточным альбумином или растворимым производным витамина Е (TPGS). Полученные формы представляют собой субмикронные суспензии с содержанием этопозида не менее 1 мг/мл: размер частиц составляет ~150 нм (в случае альбумина) и ~15 нм (в случае TPGS). Полученные формы устойчивы при лиофилизации и не содержат органических растворителей. Обе формы показали более высокую по сравнению с препаратом Этопозид-Тева цитотоксичность in vitro в отношении линии клеток опухоли молочной железы MCF-7.

Aims: To evaluate the influence of minor differences in molecular weights of commercially available low molecular weight PLGA grades on the kinetics of doxorubicin release from the nanoparticles. Methods: Three low-molecular weight 50/50 PLGA polymers were thoroughly characterised concerning intrinsic viscosity, molecular weight (Mw), acid value, and residual monomer content. The doxorubicin-loaded nanoparticles prepared using these polymers were evaluated concerning the kinetics of drug release and hydrolytic degradation. Results: The Mw of the polymers were slightly different: 10.2, 10.3, and 4.7 kDa. The nanoparticles obtained from the polymer with Mw of 4.7 kDa exhibited considerably higher rates of drug release and polymer degradation. Conclusion: In the case of low molecular weight PLGA grades even a few kilodaltons could be important for the batch-to-batch reproducibility of the nanoformulation parameters. These results bring forward the importance of in-house characterisation of the polymers to be used for the nanoparticle preparation.

PLGA (poly(lactic-co-glycolic acid))-based nanoparticles (NPs) are promising drug carrier systems because of their excellent biocompatibility and ability for sustained drug release. However, it is not well understood how the kinetics of such drug delivery system perform in the retinal blood circulation as imaged in vivo and in real time. To answer this question, PLGA NPs were loaded either with lipophilic carbocyanine perchlorate (DiI) or hydrophilic Rhodamine 123 (Rho123) and coated with poloxamer 188 (P188): PLGA-DiI/P188 and PLGA-Rho123/P188. All particles had narrow size distributions around 130 nm, spherical shape and negative potential. Subsequently, we performed in vivo real-time imaging of retinal blood vessels, combined with ex vivo microscopy to monitor the kinetics and to detect location of those two fluorescent markers. We found that DiI signals were long lasting, detectable >90 min in blood vessels after intravenous injection as visible by homogeneous labelling of the vessel wall as well as by spots in the lumen of blood vessels. In contrast, Rho123 signals mostly disappeared after 15 min post intravenous injection in such compartment. To explore how PLGA NP-loaded cargoes are released in the retina in vivo, we thereafter monitored the Cyanine5.5 amine (Cy5.5) covalently linked PLGA polymer (Cy5.5-PLGA) in parallel to DiI and Rho123. The Cy5.5 signal from PLGA polymer was detectable in the retina vessels >90 min for both, the Cy5.5-PLGA-DiI/P188 and Cy5.5-PLGA-Rho123/P188 groups. Microscopy of the ex vivo retina tissue revealed partial level of colocalization of PLGA with DiI but no colocalization between PLGA and Rho123 at 2 h post injection. This indicates that at least a fraction of the lipophilic DiI was preserved within NPs, whereas no hydrophilic Rho123 was associated with NPs at that time point. In conclusion, the properties of PLGA carrier-cargo system in the blood circulation of the retina might be strongly influenced by the combination of factors, including the individual properties of loaded compounds and blood milieu. Thus, it is unlikely that a single nanoparticle formulation will be identified that is universally effective for the delivery of different compounds.

Introduction. The use of polymeric biodegradable nanoparticles (NP) as drug delivery systems is a promising approach to overcome histohematomatic barriers. Thus, poloxamer 188-coated poly (lactide-co-glycolide) (PLGA) NP are able to overcome blood-brain barrier and to deliver therapeutic agents, in particular doxorubicin, into intracranial tumour upon intravenous administration. It is important to evaluate NP interaction with blood components in preclinical studies.The objective of the study was to investigate cytotoxicity and hemocompatibility of doxorubicin-loaded PLGA NP (Dox-PLGA NP), to essess NP uptake by glioblastoma cells.Materials and methods. The influence of NP on coagulation cascade was evaluated by prothrombin time measuring before and after plasma incubation with NP. To assess NP thrombogenicity the platelet activation level was determined by flow cytometry. The NP hemolytic activity (released hemoglobin concentration) was measured spectrophotometrically. NP cytotoxicity was determined by MTS assay. NP uptake by human glioblastoma cells was evaluated by flow cytometry.Results. Dox-PLGA NP did not influence blood coagulation time and thrombocyte activity at concentrations up to 100 mcg/mL: PT values were 12–15 s for all tested samples, and P-selectin expression level did not exceed 15 %. All samples were not hemolytic after 3 h of incubation. Cytotoxicity of doxorubicin released from PLGA NP on glioma U87MG cells was comparable to that of free doxorubicin. As shown by flow cytometry Dox-PLGA NP were efficiently internalized into the cells.Conclusion. The study of hemocompatibility confirmed the safety of Dox-PLGA NP: NP did not influence blood coagulation system and did not induce hemolysis. NP were efficiently internalized into the human glioblastoma cells and produced considerable antitumor effect in vitro.

Brain delivery of drugs by nanoparticles is a promising strategy that could open up new possibilities for the chemotherapy of brain tumors. As demonstrated in previous studies, the loading of doxorubicin in poly(lactideco-glycolide) nanoparticles coated with poloxamer 188 (Dox-PLGA) enabled the brain delivery of this cytostatic that normally cannot penetrate across the blood-brain barrier in free form. The Dox-PLGA nanoparticles produced a very considerable anti-tumor effect against the intracranial 101.8 glioblastoma in rats, thus representing a promising candidate for the chemotherapy of brain tumors that warrants clinical evaluation. The objective of the present study, therefore, was the optimization of the Dox-PLGA formulation and the development of a pilot scale manufacturing process. Optimization of the preparation procedure involved the alteration of the technological parameters such as replacement of the particle stabilizer PVA 30-70 kDa with a presumably safer low molecular mass PVA 9-10 kDa as well as the modification of the external emulsion medium and the homogenization conditions. The optimized procedure enabled an increase of the encapsulation efficiency from 66% to > 90% and reduction of the nanoparticle size from 250 nm to 110 nm thus enabling the sterilization by membrane filtration. The pilot scale process was characterized by an excellent reproducibility with very low inter-batch variations. The in vitro hematotoxicity of the nanoparticles was negligible at therapeutically relevant concentrations. The anti-tumor efficacy of the optimized formulation and the ability of the nanoparticles to penetrate into the intracranial tumor and normal brain tissue were confirmed by in vivo experiments.