Eliézer Jäger

Indomethacin-loaded nanocapsules treatment reduces in vivo glioblastoma growth in a rat glioma model

Multimodal combinations of target agents with radiation and chemotherapy may enhance cancer treatment efficacy; however, despite these treatments, gliomas recur early due to their highly proliferative, infiltrative and invasive behaviors. Nanoparticles of biodegradable polymers for anticancer drug delivery have attracted intensive interest in recent years since they may provide a sustained, controlled and targeted delivery. In the present study, we investigated the effect of indomethacin-loaded nanocapsules in an experimental glioma model. The rats treated with indomethacin-loaded nanocapsules demonstrated a significant reduction in tumor size and half of these animals presented just cells with characteristics of a residual tumor, as shown by immunostaining for nestin. Pathological analyses showed that the treated gliomas presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. An important finding of the present study is that indomethacin carried by polymeric nanocapsules achieved higher intracerebral drug concentrations than those of indomethacin in solution. Furthermore, indomethacin achieved a greater concentration in the hemisphere where the glioma was implanted, compared with the contralateral healthy hemisphere. Indomethacin-loaded nanocapsule treatment did not cause characteristics of toxicity and increased the survival of animals. Thus, our results show that polymeric nanocapsules are able to increase the intratumoral bioavailability of indomethacin and reduce the growth of implanted gliomas. Data suggest that indomethacin-loaded nanocapsules could offer new and potentially highly effective strategies for the treatment of malignant gliomas.

Effects of indomethacin-loaded nanocapsules in experimental models of inflammation in rats.

Background and purpose:  The effects of systemic treatment with indomethacin‐loaded nanocapsules (IndOH‐NC) were compared with those of free indomethacin (IndOH) in rat models of acute and chronic oedema.

pH-triggered block copolymer micelles based on a pH-responsive PDPA (poly[2-(diisopropylamino)ethyl methacrylate]) inner core and a PEO (poly(ethylene oxide)) outer shell as a potential tool for the cancer therapy

The potential of a novel pH-triggered block copolymer as a promising drug delivery platform for the cancer therapy has been explored. The block copolymer poly(ethylene oxide)-b-poly(glycerol monomethacrylate)-b-poly[2-(diisopropylamino)ethyl methacrylate] herein referred to as PEO113-b-PG2MA30-b-PDPA50 upon dissolution in ethanol followed by single-step nanoprecipitation in phosphate buffered saline (PBS) self-assembled into highly regular spherical micelles whose structure was characterized in detail by static (SLS), dynamic (DLS) and electrophoretic (ELS) light scattering, small angle X-ray scattering (SAXS), fluorescence spectroscopy and transmission electron microscopy (TEM). The micellar size (2RH = 42 nm) and micellar molecular weight (Mw(micelles) > 106 kDa) were found to be in the range to avoid renal clearance providing a long blood circulation time. Their size is below the cut-off size of the leaky pathological vasculature (DH < 200 nm), making them candidates for the use in cancer therapy based on the EPR effect. The pH-responsive PDPA core could be loaded with the poorly water-soluble anti-cancer drug paclitaxel (PTX) with encapsulation efficiency ∼70% and drug loading content ∼7% wdrug/wpolymer. The pKa of the diisopropylamino group of the PDPA block was determined as pKa = 6.8 in the simulated physiological condition, which is remarkably close to the pH microenvironment of tumoral cells. The release experiments evidenced that approximately 90% of the encapsulated PTX was sustained at the PDPA micellar core within the first 9 h at pH 7.4 whilst only 18 h were required for complete drug release at pH 5.0. These results suggest that the micellar dissociation might be triggered at the slightly acid tumoral extracellular environments (pH < pKa(PDPA)). The nanostructures were further placed in contact with human plasma or human serum albumin (HSA) diluted in PBS. The DLS experiments revealed that the micelles are especially stable for up to at least 48 h in such conditions, attesting the possibly long blood circulation time of the nanoparticles at serum environments which is a pre-requisite for the drug delivery applications. The cell viability experiments demonstrated that the drug-free block copolymer micelles are non-toxic and the number of viable cells is always greater than 85% compared to the survival number of a control group.

Lipid-core nanocapsules restrained the indomethacin ethyl ester hydrolysis in the gastrointestinal lumen and wall acting as mucoadhesive reservoirs.

The aim of this work was to investigate if the indomethacin ethyl ester (IndOEt) released from lipid-core nanocapsules (NC) is converted into indomethacin (IndOH) in the intestine lumen, intestine wall or after the particles reach the blood stream. NC-IndOEt had monomodal size distribution (242 nm; PDI 0.2) and zeta potential of -11 mV. The everted rat gut sac model showed IndOEt passage of 0.16 micromol m(-2) through the serosal fluid (30 min). From 15 to 120 min, the IndOEt concentrations in the tissue increased from 6.13 to 27.47 micromol m(-2). No IndOH was formed ex vivo. A fluorescent-NC formulation was used to determine the copolymer bioadhesion (0.012 micromol m(-2)). After NC-IndOEt oral administration to rats, IndOEt and IndOH were detected in the gastrointestinal tract (contents and tissues). In the tissues, the IndOEt concentrations decreased from 459 to 5 microg g(-1) after scrapping, demonstrating the NC mucoadhesion. In plasma (peripheric and portal vein), in spleen and liver, exclusively IndOH was detected. In conclusion, after oral dosing of NC-IndOEt, IndOEt is converted into IndOH in the intestinal lumen and wall before reaching the blood stream. The complexity of a living system was not predicted by the ex vivo gut sac model.

Selective cytotoxicity of indomethacin and indomethacin ethyl ester-loaded nanocapsules against glioma cell lines: an in vitro study.

Gliomas are the most common and devastating tumors of the central nervous system. Several studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) are promising anticancer agents. Biodegradable nanoparticulate systems have received considerable attention as potential drug delivery vehicles. The aim of this study was to evaluate the effects of indomethacin-loaded nanocapsules and indomethacin ethyl ester-loaded nanocapsules on glioma cell lines. In addition, the effect of these formulations on normal neural tissue was also evaluated. In order to investigate this, glioma cell lines (U138-MG and C6) and hippocampal organotypic cultures were used. The main finding of the present study is that indomethacin-loaded nanocapsules formulation was more potent than a solution of indomethacin in decreasing the viability and cell proliferation of glioma lines. Indomethacin and indomethacin ethyl ester associated together in the same nanocapsule formulation caused a synergic effect decreasing glioma cell proliferation. In addition, when the glioma cells were exposed to 25 microM of indomethacin-loaded nanocapsules or indomethacin ethyl ester-loaded nanocapsules, a necrotic cell death was observed. Interestingly, 5 microM of indomethacin-loaded nanocapsules was able to cause an antiproliferative effect without promoting necrosis in glioma cells. Another important finding was that the cytotoxic effect induced by 25 microM or 50 microM of indomethacin-loaded nanocapsules or indomethacin ethyl ester-loaded nanocapsules, in glioma cells was not observed in the organotypic cultures, indicating selective cytotoxicity of those formulations for tumoral cells. Further investigations using in vivo glioma model should be helpful to confirm the distinct effects of indomethacin-loaded nanocapsules and indomethacin ethyl ester-loaded nanocapsules, in normal versus tumoral cells.

Determining the simultaneous presence of drug nanocrystals in drug-loaded polymeric nanocapsule aqueous suspensions: A relation between light scattering and drug content

The encapsulation of lipophilic drugs in polymeric nanoparticles can form simultaneously both polymeric nanoparticles and drug nanocrystals. The objective was to detect the presence of nanocrystals in the nanoparticle suspensions using a simple methodology, and to determine if the nanocrystals are formed during preparation or by drug leakage from the particles during storage. Indomethacin was chosen as drug model. Unloaded and drug-loaded (1mg/mL) nanocapsules showed diameters close to 280nm and polydispersity lower than 0.20, remaining constant after 120 days. Comparing indomethacin loaded (3mg/mL) and unloaded formulations, variations in the scattered light depolarization degree indicated the simultaneous presence of nanocrystals and nanocapsules in the suspensions. A relation between the scattered light intensities and the drug precipitation was established. As a function of time, when the decrease in the Rayleigh ratios occurred, the drug contents decreased due to precipitation. On the other hand, when the Rayleigh ratios slightly increase, the drug contents are constant. The nanocrystals formed in the oversaturated formulations, agglomerate and precipitate during storage. When the drug is adsorbed on the nanocapsules, but the system is not oversaturated, no nanocrystal was formed and the formulation is physico-chemically stable at least for 150 days of storage.

Light scattering evidence of selective protein fouling on biocompatible block copolymer micelles

Selective protein fouling on block copolymer micelles with well-known potential for tumour-targeting drug delivery was evidenced by using dynamic light scattering measurements. The stability and interaction of block copolymer micelles with model proteins (BSA, IgG, lysozyme and CytC) is reported for systems featuring a hydrophobic (poly[2-(diisopropylamino)-ethyl methacrylate]) (PDPA) core and hydrophilic coronas comprising poly(ethylene oxide)/poly(glycerol monomethacrylate) (PEO-b-PG2MA) or poly[2-(methacryloyloxy)ethyl phosphorylcholine] (PMPC). The results revealed that protein size and hydrophilic chain density play important roles in the observed interactions. The PEO(113)-b-PG2MA(30)-b-PDPA(50) nanoparticles are stable and protein adsorption is prevented at all investigated protein environments. The successful protein-repellent characteristic of these nanoparticles is attributed to a high hydrophilic surface chain density (>0.1 chains per nm(2)) and to the length of the hydrophilic chains. On the other hand, although PMPC also has protein-repellent characteristics, the low surface chain density of the hydrophilic shell is supposed to enable interactions with small proteins. The PMPC(40)-b-PDPA(70) micelles are stable in BSA and IgG environments due to weak repulsion forces between PMPC and the proteins, to the hydration layer, and particularly to a size-effect where the large BSA (R(H) = 4.2 nm) and IgG (R(H) = 7.0 nm) do not easily diffuse within the PMPC shell. Conversely, a clear interaction was observed with the 2.1 nm radius lysozyme. The lysozyme protein can diffuse within the PMPC micellar shell towards the PDPA hydrophobic core in a process favored by its smaller size and the low hydrophilic PMPC surface chain density (∼0.049 chains per nm(2)) as compared to PEO-b-PG2MA (∼0.110 chains per nm(2)). The same behavior was not evidenced with the 2.3 nm radius positively charged CytC, probably due to its higher surface hydrophilicity and the consequent chemical incompatibility with PDPA.

Efficient Condensation of DNA into Environmentally Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly(ethylene oxide)113-b-poly[2-(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50), poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50), poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly{oligo(ethylene glycol)methyl ether methacrylate10-co-2-methylacrylic acid 2-[(2-(dimethylamino)ethyl)methylamino]ethyl ester44} (POEGMA70-b-P(OEGMA10-co-DAMA44). Block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (R(H) ≈ 65-85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO113-b-PDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment.

Understanding the Structural Parameters of Biocompatible Nanoparticles Dictating Protein Fouling

The development of nanocarriers for biomedical applications requires that these nanocarriers have special properties, including resistance to nonspecific protein adsorption. In this study, the fouling properties of PLA- and PCL-based block copolymer nanoparticles (NPs) have been evaluated by placing them in contact with model proteins. Block copolymer NPs were produced through the self-assembly of PEOm-b-PLAn and PEOm-b-PCLn. This procedure yielded nanosized objects with distinct structural features dependent on the length of the hydrophobic and hydrophilic blocks and the volume ratio. The protein adsorption events were examined in relation to size, chain length, surface curvature, and hydrophilic chain density. Fouling by BSA and lysozyme was considerably reduced as the length of the hydrophilic PEO-stabilizing shell increases. In contrast to the case of hydrophilic polymer-grafted planar surfaces, the current investigations suggest that the hydrophilic chain density did not markedly influence protein fouling. The protein adsorption took place at the outer surface of the NPs since neither BSA nor lysozyme was able to diffuse within the hydrophilic layer due to geometric restrictions. Protein binding is an exothermic process, and it is modulated mainly by polymer features. The secondary structures of BSA and lysozyme were not affected by the adhesion phenomena.

Soft matter assemblies as nanomedicine platforms for cancer chemotherapy: a journey from market products towards novel approaches.

The current review aims to outline the likely medical applications of nanotechnology and the potential of the emerging field of nanomedicine. Nanomedicine can be defined as the investigation area encompassing the design of diagnostics and therapeutics at the nanoscale, including nanobots, nanobiosensors, nanoparticles and other nanodevices, for the remediation, prevention and diagnosis of a variety of illnesses. The ultimate goal of nanomedicine is to improve patient quality-of-life. Because nanomedicine includes the rational design of an enormous number of nanotechnology-based products focused on miscellaneous diseases, a variety of nanomaterials can be employed. Therefore, this review will focus on recent advances in the manufacture of soft matterbased nanomedicines specifically designed to improve diagnostics and cancer chemotherapy efficacy. It will be particularly highlighted liposomes, polymer-drug conjugates, drug-loaded block copolymer micelles and biodegradable polymeric nanoparticles, emphasizing the current investigations and potential novel approaches towards overcoming the remaining challenges in the field as well as formulations that are in clinical trials and marketed products.