A range of antitumor agents for cancer treatment is available; however, they show low specificity, which often limit their use. Recently, we have reported the preparation of folate-coated long-circulating and pH-sensitive liposomes (SpHL-folate-PTX) loaded with paclitaxel (PTX), an effective drug for the treatment of solid tumors, including breast cancer. The purpose of this study was to prepare and characterize SpHL-PTX and SpHL-folate-PTX radiolabeled with technetium–99 m (99mTc). Biodistribution studies and scintigraphic images were performed after intravenous administration of 99mTc-PTX, 99mTc-SpHL-PTX and 99mTc-SpHL-folate-PTX into healthy and tumor-bearing mice. High radiochemical purity (> 98%) and in vitro stability (> 90%) were achieved for both liposome formulations. The pharmacokinetic properties of 99mTc-SpHL-DTPA-PTX and 99mTc-SpHL-folate-DTPA-PTX decreased in a monophasic manner showing half-life of 400.1 and 541.8 min, respectively. Scintigraphic images and biodistribution studies showed a significant uptake in liver, spleen and kidneys, demonstrating these routes as way for excretion. At 8 h post-injection, the liposomal tumor uptake was higher than 99mTc-PTX. Interesting, 4 h after administration, the liposome folate coated showed higher tumor-to-muscle ratio than 99mTc-SpHL-DTPA-PTX and 99mTc-PTX. In conclusion, the liposomal systems, showed high tumor uptake by scintigraphic images, especially the 99mTc-SpHL-folate-DTPA-PTX that showed a sustained and higher tumor-to-muscle ratio than non-functionalized liposome, which indicate its feasibility as a PTX delivery system to folate positive tumors.
A long-circulating and pH-sensitive liposome containing paclitaxel (SpHL-PTX) was recently developed by our group. Once in an acidic environment, for example, tumors, these liposomes undergo destabilization, releasing the encapsulated drug. In this way, the aim of this study was to evaluate the molecular and supramolecular interactions between the lipid bilayer and PTX in similar biological environment conditions. High-sensitivity analyses of SpHL-PTX structures were obtained by the small-angle X-ray scattering technique combined with other techniques such as dynamic light scattering, asymmetric flow field-flow fractionation, transmission electron microscopy, and high-performance liquid chromatography. The results showed that PTX incorporation in the liposomal bilayer clearly leads to changes in supramolecular organization of dioleoylphosphatidylethanolamine (DOPE) molecules, inducing the formation of more ordered structures. Changes in supramolecular organization were observed at lower pH, indicating that pH sensitivity was preserved even in the presence of fetal bovine serum proteins. Furthermore, morphological and physicochemical characterization of SpHL-PTX evidenced the formation of nanosized dispersion suitable for intravenous administration. In conclusion, a stable nanosized dispersion of PTX was obtained at pH 7.4 with suitable parameters for intravenous administration. At lower pH conditions, the pH sensitivity of the system was clearly evidenced by changes in the supramolecular organization of DOPE molecules, which is crucial for the delivery of PTX into the cytoplasm of the targeted cells. In this way, the results obtained by different techniques confirm the feasibility of SpHL as a promising tool to PTX delivery in acidic environments, such as tumors.
The high incidence and mortality of breast cancer supports efforts to develop innovative imaging probes to effectively diagnose, evaluate the extent of the tumor, and predict the efficacy of tumor treatments while concurrently and selectively delivering anticancer agents to the cancer tissue. In the present study we described the preparation of technetium–99 m (99mTc)-labeled paclitaxel (PTX) and evaluated its feasibility as a radiotracer for breast tumors (4T1) in BALB/c mice. Thin Layer Chromatography (TLC) was used to determine the radiochemical purity and in vitro stability of 99mTc-PTX. PTX micelles showed a unimodal distribution with mean diameter of 13.46 ± 0.06 nm. High radiochemical purity (95.8 ± 0.3%) and in vitro stability (over than 95%), up to 24 h, were observed. Blood circulation time of 99mTc-PTX was determined in healthy BALB/c mice. 99mTc-PTX decays in a one-phase manner with a half-life of 464.3 minutes. Scintigraphic images and biodistribution were evaluated at 4, 8 and 24 h after administration of 99mTc-PTX in 4T1 tumor-bearing mice. The data showed a significant uptake in the liver, spleen and kidneys, due to the importance of these routes for excretion. Moreover, high tumor uptake was achieved, indicated by high tumor-to-muscle ratios. These findings indicate the usefulness of 99mTc-PTX as a radiotracer to identify 4T1 tumor in animal models. In addition, 99mTc-PTX might be used to follow-up treatment protocols in research, being able to provide information about tumor progression after therapy.
A simple and sensitive method using first-derivative ultraviolet spectrophotometry (DS-UV) was developed, validated, and compared to the high performance liquid chromatography (HPLC) method for quantification of paclitaxel (PTX) in a liposomal formulation. Different analytical performance parameters such as linearity, accuracy, precision, specificity, detection, and quantification limits were determined according to International Conference on Harmonization (ICH) guidelines. No interference from the lipid compounds was detected in the HPLC and the DS-UV methods at 246 nm. Linearity determined for paclitaxel concentrations ranging from 6.0 to 24.0 µg mL
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presented a correlation coefficient higher than 0.999 for both methods. Relative standard deviation (RSD) values lower than 2% for intra- and inter-day precision data could be obtained. Accuracy mean values ranged from 98.9 to 102.0%. Robustness data showed that the PTX content was unaffected by the alteration proposed. Both methods were adequate to quantify the drug in the liposomal formulation. DS-UV proved to be rapid, accurate, selective, sensitive, and, therefore, an attractive tool for routine determination of PTX.
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Protein-polymer conjugates have achieved tremendous attention in the last few years, since their importance in diverse fields including drug delivery, biotechnology and nanotechnology. Over the past few years, numerous chemical strategies have been developed to conjugate different synthetic polymers onto proteins and great progress has been made. Currently, there are a handful of therapeutic polymer conjugates that have been approved by the FDA, while many hundreds of products are under extensive clinical trials and preclinical development phases. In this way, the development of novel techniques for conjugation, especially living radical polymerisation (LRP) has greatly enhanced the potential to broaden the scope of therapeutic conjugates. As a consequence, versatile techniques have developed, such as the 'grafting from' approach, which allows modifications of biomacromolecules at the atomic level, and subsequently preparing well-defined stimuli-responsive conjugates. These strategies present a unique perspective for therapy expansion of a new generation of 'smart' products with proprieties that can be finely controlled and tuned rather than just enhanced. This article highlights recent advances in the synthesis and application of protein-polymer conjugates by controlled radical polymerisation techniques, with special emphasis on stimuli-responsive conjugates on new applications in biomedical and pharmaceutical areas.
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