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Atomic force microscopy (AFM) visualization and analysis of nanocarriers. Curcumin-loaded nanocarriers produced by the suspension method are deposited on mica surface and visualized with AFM (A). To evaluate the size distribution of the produced nanocarriers, deposition with a lower coverage was obtained (B), where the particles are well separated and can be identified and measured using WSxM flooding procedure (C). The resulting size distribution obtained after processing of ten different low-coverage images is shown (D).
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Aim:
Ability to deliver drugs into the cell nuclei can significantly increase the efficacy of cancer therapies, in particular in the case of multidrug-resistant cancer Results: Polymer nanocarriers based on amphiphilic thiooctadecyl-terminated poly-N-vinyl-2-pyrrolidone were produced and loaded with a model hydrophobic drug, curcumin. Two commonly...
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... size distribution of curcumin-loaded nanocarriers was further visualized and evaluated using AFM. A representative topographic image showing nanocarriers deposited on mica at high density is shown in Figure 4A. To evaluate the size distribution, a lower density deposition was made and ten different images similar to Figure 4B were analyzed. ...
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... representative topographic image showing nanocarriers deposited on mica at high density is shown in Figure 4A. To evaluate the size distribution, a lower density deposition was made and ten different images similar to Figure 4B were analyzed. Each image was processed using the flooding procedure of WSxM to determine the center of each micelle and its height, as illustrated in Figure 4C. ...
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... evaluate the size distribution, a lower density deposition was made and ten different images similar to Figure 4B were analyzed. Each image was processed using the flooding procedure of WSxM to determine the center of each micelle and its height, as illustrated in Figure 4C. This yielded a distribution shown in Figure 4D with a mean height of 2.7 nm and SD of 0.9 nm. ...
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... image was processed using the flooding procedure of WSxM to determine the center of each micelle and its height, as illustrated in Figure 4C. This yielded a distribution shown in Figure 4D with a mean height of 2.7 nm and SD of 0.9 nm. It can be seen that the micelles collapse when deposited on the surface and the hydrodynamic diameter of the nanocarriers determined by NTA is significantly larger than the size of collapsed micelles visualized by AFM. ...
Citations
... The impact of PVP onto the fluorescence lifetime was demonstrated by the data obtained in solution (Fig. 3). Since passive diffusion is a proposed cell uptake pathway for small NPs including PVP (MW of 10 kDa applied here) [81], PVP loaded particles are likely to reside in the plasma membrane. Only a slow release into the cytoplasm may lead to the appearance of short-lived components in the cytoplasm and an overall spread after 24 h. ...
... Cell internalization of micelles and PVP takes place via passive diffusion and/or via the endocytic pathway (Fig. 7B). The size of the carriers can strongly affect the cellular uptake pathway and small particles including PVP with a MW of 10 kDa used in this study are capable to enter the cell via passive diffusion [81]. For the less rigid micelles, several cell entry routes may coexist including direct drug release into the plasma membrane (Fig. 7B) [82,83]. ...
... Cell uptake assay was performed on A431 and HEK 293 cell lines according to our previous work [27]. Briefly, the cells were seeded in a 96-well polystyrene plate at a density of 5 × 10 3 cells per well. ...
... The molecular weight of the resulting polymer was 6 kDa. As it was shown previously, this molecular weight accounts for suitable critical aggregation concentration and low toxicity of the polymer [21,25,27]. PVP-Cur NPs were obtained by the solvent evaporation method: dispersion was carried out using an ultrasonic homogenizer using two infinitely compatible solvents (acetone/water). ...
Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was −4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.
... They can capture various therapeutic agents, such as nonsteroidal anti-inflammatory drugs [19][20][21], antitumor drugs [22], antifungal antibiotics [23], cytokines [24,25] and plasmid DNA [26] into micelles. Depending on the average hydrodynamic radius, nanoparticles can be absorbed by cell cultures through various mechanisms, including endocytosis and membrane fusion, which allows selective delivery of the active substance to both cell endosomes and its nucleus [27]. In addition, PVP nanoparticles can be used as effective modifiers of liposomal membranes, increasing their stability [23]. ...
... The synthesis of amphiphilic poly(N-vinylpyrrolidone) with a molecular weight of 6 kDa was carried out in accordance with [18,27,43]. Briefly, 20 ml of N-vinylpyrrolidone, AIBN 1 wt% (0,7 mol%) and ODM 0,25 wt% (1 mol%) were dissolved in 40 ml of 1,4-dioxane. ...
... Cell uptake assay was studied on A431 and HEK 293 cell lines according to our previous works [27]. Briefly, the cells were seeded in a 96-well polystyrene plate at a density of 5×10 3 cells per well. ...
Curcumin attracts a huge attention because of its biological properties: antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the suspension method and were characterized by dynamic and electrophoretic light scattering, transmission electron microscopy and atomic force microscopy. The average particle size was 200 nm, and ζ-potential was -4 mV. Curcumin release studies showed that nanoparticles are stable in water solution. In vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to pure curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. Median lethal dose was determined in acute toxicity assay on Zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.
... While PVP may not be internalized, only releasing its payload into the HeLa cells, this assumption is rather unlikely according to literature reports on the cellular uptake of NPs. As an example, it could be shown that PVP covalently bound to hypericin was internalized into HeLa cells [66], and for octadecyl-modified PVP NPs, dynamin-dependent endocytosis was suggested as a major uptake mechanism besides passive diffusion through the cell membrane [65,67,68]. Accordingly, it is most likely in the present work that intracellular PVP is associated with cellular macromolecular structures like proteins or membranes, further increasing the line-broadening of the PVP resonances, leading to their NMR-invisibility. ...
Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is affected by nanoparticles. In a systematic study, high-resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle-forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion-edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration-dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP, whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA, and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration-dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline the important protective role of the polymeric carriers following cell internalization. Moreover, to our knowledge, for the first time, the current study allowed us to trace intracellular PS localization on an atomic level by NMR methods.
... While PVP may not be internalized but only release its payload into the HeLa cells, this assumption is rather unlikely according to literature reports on cellular uptake of NPs. As an example it could be shown that PVP covalently bound to hypericin was internalized into HeLa cells [66] and for octadecyl-modified PVP NPs, dynamindependent endocytosis was suggested as major uptake mechanism besides passive diffusion through the cell membrane [65,67,68]. Accordingly, it is most likely in the present work that intracellular PVP is associated with cellular macromolecular structures like proteins or membranes further increasing the line-broadening of the PVP resonances leading to their NMR-invisibility. ...
Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is af-fected by nanoparticles. In a systematic study, high resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of the PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP, but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline an important protective role of the polymeric carriers following cell internalization. Moreover, the current study allowed - to our knowledge for the first time - to trace the intracellular PS localization on an atomic level by NMR methods.
... The main cell uptake routes for nanoparticles are not only via the endocytic pathway but also via passive diffusion. 72,73 Intracellular redistribution and interactions with cellular components, like proteins, will affect the fluorescence decay dynamics in the cellular environment. When applied as Pc1-PVP, Pc1 exhibited a similar fluorescence lifetime distribution as in solution, which indicates that the phthalocyanine may remain associated with the PVP carrier inside the cells. ...
Phthalocyanines are ideal candidates as photosensitizers for photodynamic therapy (PDT) of cancer due to their favorable chemical and photophysical properties. However, their tendency to form aggregates in water reduces PDT efficacy and poses challenges in obtaining efficient forms of phthalocyanines for therapeutic applications. In the current work, polyvinylpyrrolidone (PVP) and micellar formulations were compared for encapsulating and monomerizing a water-soluble zinc phthalocyanine bearing four non-peripheral triethylene glycol chains (Pc1). 1H NMR spectroscopy combined with UV-vis absorption and fluorescence spectroscopy revealed that Pc1 exists as a mixture of regioisomers in monomeric form in dimethyl sulfoxide but forms dimers in an aqueous buffer. PVP, polyethylene glycol castor oil (Kolliphor RH40), and three different triblock copolymers with varying proportions of polyethylene and polypropylene glycol units (termed P188, P84, and F127) were tested as micellar carriers for Pc1. 1H NMR chemical shift analysis, diffusion-ordered spectroscopy, and 2D nuclear Overhauser enhancement spectroscopy was applied to monitor the encapsulation and localization of Pc1 at the polymer interface. Kolliphor RH40 and F127 micelles exhibited the highest affinity for encapsulating Pc1 in the micellar core and resulted in intense Pc1 fluorescence emission as well as efficient singlet oxygen formation along with PVP. Among the triblock copolymers, efficiency in binding and dimer dissolution decreased in the order F127 > P84 > P188. PVP was a strong binder for Pc1. However, Pc1 molecules are rather surface-attached and exist as monomer and dimer mixtures. The results demonstrate that NMR combined with optical spectroscopy offer powerful tools to assess parameters like drug binding, localization sites, and dynamic properties that play key roles in achieving high host-guest compatibility. With the corresponding adjustments, polymeric micelles can offer simple and easily accessible drug delivery systems optimizing phthalocyanines' properties as efficient photosensitizers.
... Recently, the authors developed an approach to the synthesis of linear amphiphilic polymers based on low-molecular-weight poly(N-vinyl-2-pyrrolidone) with hydrophilic alkyl end groups. The amphiphilic low-molecular-weight poly(N-vinyl-2-pyrrolidone) proved to be effective in terms of loading with hydrophobic substances [20,21] and does not display any significant toxicity [22,23]. However, the amphiphilic N-vinyl-2-pyrrolidone homopolymer is relatively ineffective as a carrier for drugs with high solubility in water [24], which limits the possibilities of its application in combination therapy. ...
... Numerous studies show that due to the high hydrophobicity and negligible solubility of paclitaxel in water, the process of its release from the hydrophobic cores of nanosized aggregates takes days [62][63][64]. We recently showed that the uptake by U87 and CRL 2429 cells of aggregates of a structurally similar amphiphilic N-vinyl-2-pyrrolidone homopolymer loaded with a hydrophobic DiI dye is very efficient and reaches saturation within few hours [20,21]. Given the high uptake rate of amphiphilic poly(N-vinyl-2-pyrrolidone) aggregates by cells, paclitaxel appears to be involved in metabolic processes prior to its passive release from nanoscale aggregates via diffusion. ...
Co-delivery of chemotherapeutics in cancer treatment has been proven essential for overcoming multidrug resistance and improving the outcome of therapy. We report the synthesis of amphiphilic copolymers of N-vinyl-2-pyrrolidone and allyl glycidyl ether of various compositions and demonstrate that they can form nanoaggregates capable of simultaneous covalent immobilization of doxorubicin by the epoxy groups in the shell and hydrophobic-driven incorporation of paclitaxel into the core of nanoparticles. The structure of the obtained copolymers was characterized by 13C NMR, IR, and MALDI spectroscopy, as well as adsorption at the water/toluene interface. A linear increase in the number-average molecular weight of amphiphilic copolymers and a decrease in the number-average diameter of macromolecular aggregates with an increase in the ratio N-vinyl-2-pyrrolidone/allyl glycidyl ether were observed. The assembled nanocarriers were characterized by DLS. The reported novel nanocarriers can be of interest for delivery and co-delivery of a wide range of pharmacological preparations and combined therapy for cancer and other deceases.
... Fullerenes, nanotubes [1,2] and other non-biological nanomaterials modified with moieties increasing biocompatibility, e.g., polyethylene glycol, are being studied as potential drug delivery platforms. In many works, polymer micelles with a size of 10-100 nm were demonstrated to be suitable nanocontainers for drugs [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. For preparation of polymer micelles in solution, amphiphilic block copolymers consisting of biodegradable hydrophobic polymer blocks such as polyesters or polyamino acids and biocompatible hydrophilic blocks such as polyethylene glycol, poly-Nvinyl-2-pyrrolidone, poly-2-ethyl-2-oxazoline or polyacrylic acid have been widely used [10]. ...
... They have been shown to exhibit low toxicity, their safety was demonstrated both in vitro and in vivo, and they are able to deliver an active agent into the cell and increase the bioavailability of hydrophobic drugs. At present, micellar particles of amphiphilic polymers of N-vinylpyrrolidone have been proposed as delivery systems for antibiotics [14][15][16][17][18][19]. ...
New amphiphilic terpolymers of N-vinylpyrrolidone, poly(ethylene glycol) methyl ether methacrylate (Mn 500 g/mol) and triethylene glycol dimethacrylate (branching agent) were synthesized via radical polymerization with and without 1-decanethiol as a chain transfer agent. They consisted of ca. 10 mol% (di)methacrylate units and had effective absolute molecular weight of about 26–600 kDa. The terpolymers were studied in polar media with dynamic light scattering, and critical concentration of aggregation and sizes of individual macromolecules and their aggregates were determined. Nanostructures of terpolymers containing 0.25–0.75% of fluorescent zinc tetraphenylporphyrinate (ZnTPP) were obtained that had the hydrodynamic radii less than 100 nm in aqueous solutions. The molar extinction coefficient of the Soret band at 425 nm of the encapsulated ZnTPP and the effective terpolymer binding constant for ZnTPP and the stability of terpolymers were found to be dependent on the presence of hydrophobic –SC10H21 groups in terpolymer chains. Terpolymer 1 did not affect significantly the viability of Vero or HeLa cells and was able to penetrate cells with high efficiency. Thus, new non-toxic amphiphilic terpolymers of N-vinylpyrrolidone can be promising platforms for delivery of hydrophobic biologically active compounds.
Graphic abstract
... The nanoaggregates of N-vinyl-2-pyrrolidone oligomers with a terminal thiooctadecyl group (PVP-OD) have been shown to form particles of 50 nm size capable of transporting curcumin into cell nuclei [23]. At the same time, the uptake mechanism and kinetics of the aggregates of PVP-OD ranging in size from 100 nm to 500 nm, which are usually formed because of self-assembly of chains without fractionation, remain unknown. ...
... Therefore, it can be assumed that DiI is incorporated into the hydrophobic regions of aggregates formed by the octadecyl sections of amphiphilic PVP-OD, hence changing just the size of the aggregates and not their morphology. It should be noted that although number of works have addressed drug loading into nanosized aggregates based on amphiphilic PVP [22,23], the conservation of the overall morphology and the number of particles accompanied by an increase in their average size upon drug loading has not been shown earlier. Nano-tracking analysis of PVP-OD aggregates with a number average molecular mass of ̅ = 6000 in water shows formation of aggregates with an average diameter of 105 nm and a mode of 37 nm was observed. ...
... Human primary foreskin fibroblasts (CRL 2429) and the human glioblastoma cell line (U87) were obtained from American Type Culture Collection (ATCC). Cells were grown in Dulbecco's modified Eagle Medium (DMEM F12, Thermo Scientific Fisher) supplemented with 100 U/mL penicillin, 10 mg/mL streptomycin, and 10% FBS in a humid environment, 5% CO 2 at 37 • C [23]. The cells were then seeded in 96 well plates so that the cell density was about 5 × 10 3 cells × cm −2 . ...
Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier–membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to M¯n = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.
... Amph-PVP-based nanocarriers were proposed for the delivery of non-steroidal antiinflammatory drugs [39,40] and for drug delivery into the cell nucleus [41]. Moreover, amphiphilic linear-dendritic block copolymers based on the linear PVP and dendritic phenylalanyl-lysine dipeptide were reported for the enzyme-responsive release of doxoru-bicin [42]. ...
