Poly-ε-caprolactone based nanoparticles for delivery of genistein in melanoma treatment

We developed poly-ε-caprolactone (PCL)-based nanoparticles containing D-α-tocopherol polyethylene glycol-1000 succinate (TPGS) or Poloxamer 407 as stabilizers to efficiently encapsulate genistein (GN). Two formulations, referred to as PNTPGS and PNPol, were prepared using nanoprecipitation. They were characterized by size and PDI distribution, zeta potential, nanoparticle tracking analysis (NTA), GN association (AE%), infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). PNTPGS-GN exhibited a particle size of 141.2 nm, a PDI of 0.189, a zeta potential of -32.9 mV, and an AE% of 77.95%. PNPol-GN had a size of 146.3 nm, a better PDI than PNTPGS-GN (0.150), a less negative zeta potential (-21.0 mV), and an AE% of 68.73%. Thermal and spectrometric analyses indicated that no new compounds were formed, and there was no incompatibility detected in the formulations. Cellular studies revealed that Poloxamer 407 conferred less toxicity to PCL nanoparticles. However, the percentage of uptake decreased compared to the use of TPGS, which exhibited almost 80% cellular uptake. This study contributes to the investigation of stabilizers capable of conferring stability to PCL nanoparticles efficiently encapsulating GN. Thus, the PCL nanoparticle proposed here is an innovative nanomedicine for melanoma therapy and represents a strong candidate for specific pre-clinical and in vivo studie

Epirubicin: Biological Properties, Analytical Methods, and Drug Delivery Nanosystems.

Epirubicin (EPI) is a chemotherapeutic agent belonging to the anthracycline drug class indicated for treating several tumors. It acts by suppressing the DNA and RNA synthesis by intercalating between their base pair. However, several side effects are associated with this therapy, including cardiotoxicity and myelosuppression. Therefore, EPI delivery in nanosystems has been an interesting strategy to overcome these limitations and improve the safety and efficacy of EPI. Thus, analytical methods have been used to understand and characterize these nanosystems, including spectrophotometric, spectrofluorimetric, and chromatography. Spectrophotometric and spectrofluorimetric methods have been used to quantify EPI in less complex matrices due to their efficiency, low cost, and green chemistry character. By contrast, high-performance liquid chromatography is a suitable method for detecting EPI in more complex matrices (e.g., plasm and urine) owing to its high sensitivity. This review summarizes physicochemical and pharmacokinetic properties of EPI, its application in drug delivery nanosystems, and the analytical methods employed in its quantification in different matrices, including blood, plasm, urine, and drug delivery nanosystems.

Targeted Liposomes: A Nonviral Gene Delivery System for Cancer Therapy.

Cancer is the second most frequent cause of death worldwide, with 28.4 million new cases expected for 2040. Despite de advances in the treatment, it remains a challenge because of the tumor heterogenicity and the increase in multidrug resistance mechanisms. Thus, gene therapy has been a potential therapeutic approach owing to its ability to introduce, silence, or change the content of the human genetic code for inhibiting tumor progression, angiogenesis, and metastasis. For the proper delivery of genes to tumor cells, it requires the use of gene vectors for protecting the therapeutic gene and transporting it into cells. Among these vectors, liposomes have been the nonviral vector most used because of their low immunogenicity and low toxicity. Furthermore, this nanosystem can have its surface modified with ligands (e.g., antibodies, peptides, aptamers, folic acid, carbohydrates, and others) that can be recognized with high specificity and affinity by receptor overexpressed in tumor cells, increasing the selective delivery of genes to tumors. In this context, the present review address and discuss the main targeting ligands used to functionalize liposomes for improving gene delivery with potential application in cancer treatment.

Highlights in targeted nanoparticles as a delivery strategy for glioma treatment.

Glioma is the most common type of Central Nervous System (CNS) neoplasia and it arises from glial cells. As glial cells are formed by different types of cells, glioma can be classified according to the cells that originate it or the malignancy grade. Glioblastoma multiforme is the most common and aggressive glioma. The high lethality of this tumor is related to the difficulty in performing surgical removal, chemotherapy, and radiotherapy in the CNS. To improve glioma treatment, a wide range of chemotherapeutics have been encapsulated in nanosystems to increase their ability to overcome the blood-brain barrier (BBB) and specifically reach the tumoral cells, reducing side effects and improving drug concentration in the tumor microenvironment. Several studies have investigated nanosystems covered with targeting ligands (e.g., proteins, peptides, aptamers, folate, and glucose) to increase the ability of drugs to cross the BBB and enhance their specificity to glioma through specific recognition by receptors on BBB and glioma cells. This review addresses the main targeting ligands used in nanosystems to overcome the BBB and promote the active targeting of drugs for glioma. Furthermore, the advantages of using these molecules in glioma treatment are discussed.

Docetaxel-loaded folate-modified TPGS-transfersomes for glioblastoma multiforme treatment.

Glioblastoma multiforme (GBM) is a first primary Central Nervous System tumor with high incidence and lethality. Its treatment is hampered by the difficulty to overcome the blood-brain barrier (BBB) and by the non-specificity of chemotherapeutics to tumor cells. This study was based on the development characterization and in vitro efficacy of folate-modified TPGS transfersomes containing docetaxel (TF-DTX-FA) to improve GBM treatment. TF-DTX-FA and unmodified transfersomes (TF-DTX) were prepared through thin-film hydration followed by extrusion technique and characterized by physicochemical and in vitro studies. All formulations showed low particles sizes (below 200 nm), polydispersity index below 0.2, negative zeta potential (between -16.75 to -12.45 mV) and high encapsulation efficiency (78.72 ± 1.29% and 75.62 ± 0.05% for TF-DTX and TF-DTX-FA, respectively). Furthermore, cytotoxicity assay of TF-DTX-FA showed the high capacity of the nanocarriers to reduce the viability of U-87 MG in both 2D and 3D culture models, when compared with DTX commercial formulation and TF-DTX. In vitro cellular uptake assay indicated the selectivity of transfersomes to tumoral cells when compared to normal cells, and the higher ability of TF-DTX-FA to be internalized into 2D U-87 MG in comparison with TF-DTX (72.10 and 62.90%, respectively, after 24 h). Moreover, TF-DTX-FA showed higher permeability into 3D U-87 MG spheroid than TF-DTX, suggesting the potential FA modulation to target treatment of GBM.

Generation of a Three-Dimensional in Vitro Ovarian Cancer Co-Culture Model for Drug Screening Assays.

In vitro 3D culture models have emerged in the cancer field due to their ability to recapitulate characteristics of the in vivo tumor. Herein, we described the establishment and characterization of 3D multicellular spheroids using ovarian cancer cells (SKOV-3) in co-culture with mesenchymal cells (MUC-9) or fibroblasts (CCD27-Sk). We demonstrated that SKOV-3 cells in co-culture were able to form regular and compact spheroids with diameters ranging from 300 to 400 µm and with a roundness close to 1.0 regardless of the type of stromal cell used. In the 3D culture an increase was not observed in spheroid diameter nor was there significant cell growth. What is more, the 3D co-cultures presented an up regulation of genes related to tumorigenesis, angiogenesis and metastases (MMP2, VEGFA, SNAI1, ZEB1 and VIM) when compared with 2D and 3D monoculture. As expected, both 3D cultures (mono and co-cultures) exhibited a higher Paclitaxel chemoresistance when compared to 2D condition. Although we did not observe differences in the Paclitaxel resistance between the 3D mono and co-cultures, the gene expression results indicate that the presence of mesenchymal cells and fibroblasts better recapitulate the in vivo tumor microenvironment, being able, therefore, to more accurately evaluate drug efficacy for ovarian cancer therapy.

Gene Therapy Based on Lipid Nanoparticles as Non-viral Vectors for Glioma Treatment.

Gliomas are primary brain tumors originating from glial cells, representing 30% of all Central Nervous System (CNS) neoplasia. Among them, the astrocytoma grade IV (glioblastoma multiforme) is the most common, presenting an invasive and aggressive profile, with an estimated life expectancy of about 15 months after diagnosis even after treatment with radiation, surgical resection, and chemotherapy. This poor prognosis is related to the presence of the blood-brain barrier (BBB) and multidrug resistance mechanisms that prevent the uptake and retention of chemotherapeutics inside the brain. Gene therapy has been a promising strategy to overcome these treatment limitations since it has the ability to modify the defective genetic information in tumor cells, being able to induce cellular apoptosis and silence the genes responsible for multidrug resistance. Lipidbased nanoparticles, non-viral vectors, have been investigated to deliver genes across the BBB to reach the glioma cell target. Besides, their low immunogenicity, easy production, ability to incorporate ligands to specific target cells, and capacity to carry higher size genes have made the gene therapy based on non-viral vectors a promising glioma treatment. In this context, this review addresses the most common non-viral vectors based on lipid-based nanoparticles used for glioma gene therapy, such as liposomes, solid lipid nanoparticles, nanostructured lipid carriers, and nanoemulsions.

EGFR-targeted immunoliposomes efficiently deliver docetaxel to prostate cancer cells.

Prostate cancer is the second cause of cancer death in men worldwide. Docetaxel (DTX), an antimitotic drug, is widely used for the treatment of metastatic prostate cancer patients. Taxotere® is a commercial DTX formulation. It contains a polysorbate 80 surfactant to improve DTX aqueous solubility, which has been associated with hypersensitivity reactions in patients. Liposomes have been used as promising delivery systems for a range of hydrophobic drugs, such as DTX, offering improved drug water solubility and biocompatibility, without compromising its anticancer activity. Herein, DTX-loaded liposomes were developed using the Box-Behnken factorial design. The optimized formulation was nano-sized, homogenous in size (67.47 nm) with high DTX encapsulation efficiency (99.95 %). The encapsulated DTX was in a soluble amorphous state, which was slowly released. Next, to increase the liposomes selectivity to prostate cancer cells, cetuximab, an anti-EGFR monoclonal antibody. was successfully conjugated to the surface of liposomes, without compromising cetuximab protein structure and stability. As expected, our results showed higher cellular uptake and toxicity of immunoliposomes, compared to non-targeted liposomes, in DU145 (EGFR-overxpressing) prostate cancer cells. To the best of our knowledge, this is the first report of engineering EGFR-targeted liposomes to enhance the selectivity of DTX delivery to EGFR-positive prostate cancer cells.

In vitro evaluation of folate-modified PLGA nanoparticles containing paclitaxel for ovarian cancer therapy.

Ovarian cancer is the most lethal gynecological cancer of female reproductive system. In order to improve the survival rate, some modifications on nanoparticles surfaces have been investigated to promote active targeting of drugs into tumor microenvironment. The aim of this study was the development and characterization of folate-modified (PN-PCX-FA) and unmodified PLGA nanoparticles (PN-PCX) containing paclitaxel for ovarian cancer treatment. Nanocarriers were produced using nanoprecipitation technique and characterized by mean particle diameter (MPD), polydispersity index (PDI), zeta potential (ZP), encapsulation efficiency (EE), DSC, FTIR, in vitro cytotoxicity and cellular uptake. PN-PCX and PN-PCX-FA showed MPD < 150 nm and PDI < 0.2 with high EE (about 90%). Cytotoxicity assays in SKOV-3 cells demonstrated the ability of both formulations to cause cellular damage. PCX encapsulated in PN-PCX-FA at 1 nM showed higher cytotoxicity than PN-PCX. Folate-modified nanoparticles showed a 3.6-fold higher cellular uptake than unmodified nanoparticles. PN-PCX-FA is a promising system to improve safety and efficacy of ovarian cancer treatment. Further in vivo studies are necessary to prove PN-PCX-FA potential.

Development, characterization and biological in vitro assays of paclitaxel-loaded PCL polymeric nanoparticles.

Adenocarcinoma is the most lethal gynecologic tumor and treatment usually consists in surgery followed by chemotherapy. However, the chemotherapy benefits are eventually limited due to drug toxicity to normal tissues and cells, which cause several and harsh side effects. Paclitaxel (PCX) is the drug of first choice for ovarian cancer treatment, but it has low aqueous solubility, which reduces its bioavailability. Thus, in the commercial drug, Taxol®, PCX is solubilized in a mixture of toxic surfactants. The development of drug nanocarriers has been investigated to promote the reduction of toxic effects and increase the safety and therapeutic efficacy of PCX. The aim of this work was the development and characterization of PCX loaded nanoparticles (PNPCX) and evaluation of in vitro efficacy of developed system using adenocarcinoma cell line. The nanocarrier was successfully obtained using nanoprecipitation technique. The results showed that the PNPCX-A had a particle size distribution around 140 nm and polydispersity index smaller than 0.1, with high PCX encapsulation efficiency. The results obtained were suitable for the intravenous administration route and promotion of passive targeting in the tumor microenvironment. The in vitro cytotoxicity assays of SKOV-3 cell line demonstrated that PNPCX-A was able to release PCX and reduce cell viability. The flow cytometry assays first reported that a nanostructured system with such composition (PNPCX-A) presented a time dependent cellular uptake, showing the ability of nanocarrier to be internalized. PNPCX-A present a distinguish potential for ovarian cancer therapy optimization. In vivo studies are needed to confirm the in vitro results and provide additional data regarding safety and efficacy of ovarian cancer treatment.

Description of the complexity of prescribed medication regimens in primary health care of Ribeirão Preto - SP

Introduction: Pharmacotherapy is the main therapeutic resource for the management of diseases. However, the number of drugs prescribed, dose frequency, and mode of administration can make the treatment more complex and influence treatment outcomes. The aim of this study was to measure the complexity of prescribed medication regimens in primary health care (PHC) services in Ribeirão Preto, Brazil. Methods: This cross-sectional study included 1,009 participants: 889 from primary health units and 120 from family health units in Ribeirão Preto, Brazil. Treatment complexity was assessed using the Medication Regimen Complexity Index (MRCI). Results: MRCI mean scores were 12.5 points (SD = 9.3) and dose frequency was the major contributor to increase the score. The complexity of pharmacotherapy showed a significant correlation with the number of prescribed medications (r = 0.93, p < 0.01), but not with patients' age (r = 0.28, p < 0.01). There is also no difference in complexity between the sexes (p = 0.83) and the types of primary health care service (p = 0.31). An analysis of variance revealed that patients with lower levels of education receive more complex prescriptions (p < 0.01). Conclusions: The pharmacotherapy prescribed in PHC services from Ribeirão Preto, Brazil is complex, and there is a need to concentrate efforts and adopt strategies to simplify drug prescription without compromising patient's clinical status.

Adequação da prescrição de medicamentos na Atenção Primária à Saúde de Ribeirão Preto-SP: estudo transversal / Adequacy of drug prescriptions in the Primary Health Care of Ribeirão Preto-SP: a cross-sectional study / Adecuación de las prescripciones de medicamentos en la Atención Primaria de Salud de Ribeirão Preto-SP: estudio transversal

Objetivo: Este estudo transversal visa analisar comparativamente as prescrições de medicamentos provenientes da atenção básica tradicional (Unidades Básicas de Saúde - UBS) com as da Estratégia de Saúde da Família (ESF). Métodos: Foram incluídas 1053 prescrições, alocadas em dois grupos: 932 provenientes de UBS e 121 da ESF. Tais prescrições foram analisadas de acordo com a adequação aos itens legalmente exigidos e aos indicadores de qualidade (presença de antimicrobianos, presença de injetáveis, uso da denominação oficial, uso da relação de medicamentos essenciais, média de medicamentos prescritos). Resultados: As prescrições da ESF se mostraram estatisticamente mais completas quanto à presença do endereço do prescritor (82,6% UBS, 96,7% ESF), à ausência de rasuras (90,3% UBS, 96,7% ESF) e ao cumprimento dos aspectos legais exigidos referentes ao uso do medicamento, sendo eles: forma farmacêutica (70,7% UBS, 80,2% ESF), dose (70,9% UBS, 79,3% ESF), posologia (63,0% UBS, 75,2% ESF), via de administração (58,3% UBS, 83,5% ESF) e duração do tratamento (76,9% UBS, 92,6% ESF). Os resultados dos indicadores de qualidade da prescrição se aproximaram nos dois modelos de atenção básica e estão de acordo com os valores recomendados, com exceção da média de medicamentos prescritos, a qual evidenciou uma tendência à polifarmácia no município (3,9 medicamentos por prescrição na UBS e 3,5 na ESF). Conclusão: Apesar dos resultados revelarem práticas inapropriadas na prescrição de medicamentos na Atenção Primária como um todo, há evidências de que as prescrições da ESF estão mais próximas ao padrão ideal, o que pode favorecer o Uso Racional de Medicamentos.

Objective: This cross-sectional study aims to comparatively analyze drug prescriptions from the traditional basic health care service (Basic Health Units - BHU) with those from Family Health Strategy (FHS). Methods: A total of 1053 prescriptions were included, allocated in two groups: 932 from BHU and 121 from FHS. These prescriptions were analyzed according with compliance to legally required items and quality indicators (presence of antimicrobials, presence of injectable, use of the official name, use of the list of essential drugs, average of drugs prescribed). Results: The ESF prescriptions were more complete regarding the presence of the prescriber's address (82.6% BHU, 96.7% FHS), the absence of erasures and overwritten words (90.3% BHU, 96.7% FHS) and with compliance to legally required items related to use of drugs, being: pharmaceutical form (70.7% BHU, 80.2% FHS), dose (70.9% BHU, 79.3% FHS), posology (63.0% BHU, 75.2% FHS), administration route (58.3% BHU, 83.5% FHS) and time of therapy (76.9% BHU, 92.6% FHS). The results of the indicators of quality of prescription are approximated in both models of basic attention and according to the recommended values, with the exception of the average of drugs prescribed, which showed a tendency to polypharmacy at county (3.9 drugs per prescription at the BHU and 3.5 at the FHS). Conclusion: although the results reveal inappropriate practices in drug prescription at Primary Health Care services as a whole, there are evidence that prescriptions from FHS are closer to the ideal pattern, which may favor the Rational Use of Drugs.

Objetivo: Este estudio transversal tuvo como objetivo comparar prescripciones médicas de la atención primaria tradicional (Unidades Básicas de Salud - UBS) con las de la Estrategia de Salud Familiar (ESF). Métodos: Se incluyeron 1053 prescripciones, divididas en dos grupos: 932 de la UBS y 121 de la ESF. Estas prescripciones se analizaron de acuerdo con la adecuación de los ítems legalmente requeridos y a los indicadores de calidad (presencia de antimicrobianos, presencia de inyectables, uso de la denominación oficial, uso de la relación de medicamentos esenciales, número medio de los medicamentos prescritos). Resultados: Las prescripciones de la ESF eran estadísticamente más completas debido a la presencia de la dirección del prescriptor (82,6% UBS, 96,7% ESF), en la ausencia de tachaduras (90,3% UBS, 96,7% ESF) y en el cumplimiento de los aspectos legales requeridos referentes al uso del medicamento, siendo ellos: forma farmacéutica (70,7% UBS, 80,2% ESF), dosis (70,9% UBS, 79,3% ESF), posología (63,0% UBS, 75,2% ESF), vía de administración (58,3% UBS, 83,5% ESF) y duración del tratamiento (76,9% UBS, 92,6% ESF). Los resultados de los indicadores de calidad de la prescripción fueron similares en los dos modelos de atención primaria y están de acuerdo con los valores recomendados, excepto el número medio de los medicamentos prescriptos, que mostró una tendencia a la polifarmacia en el municipio (3,9 medicamentos por prescripción en la UBS y 3,5 en la ESF). Conclusión: Aunque los resultados indican prácticas inadecuadas en la prescripción de medicamentos en atención primaria en general, existen evidencias de que las prescripciones de la ESF son más cercanas a los estándares, lo que puede favorecer el Uso Racional de Medicamentos.

Poly-epsilon-caprolactone nanoparticles enhance ursolic acid in vivo efficacy against Trypanosoma cruzi infection.

Despite affecting millions of people worldwide, Chagas disease is still neglected by the academia and industry and the therapeutic option available, benznidazole, presents limited efficacy and side effects. Within this context, ursolic acid may serve as an option for treatment, however has low bioavailability, which can be enhanced through the encapsulation in polymeric nanoparticles. Therefore, herein we developed ursolic acid-loaded nanoparticles with poly-ε-caprolactone by the nanoprecipitation method and characterized them for particle size, zeta potential, polydispersity, encapsulation efficiency, morphology by scanning electron microscopy and thermal behavior by differential scanning calorimetry. Results indicated that an appropriate ratio of organic phase/aqueous phase and polymer/drug is necessary to produce smaller particles, with low polydispersity, negative zeta potential and high drug encapsulation efficiency. In vitro studies indicated the safety of the formulation against fibroblast culture and its efficacy in killing T. cruzi. Very importantly, the in vivo study revealed that the ursolic acid-loaded nanoparticle is as potent as the benznidazole group to control parasitemia, which could be attributed to improved bioavailability of the encapsulated drug. Finally, the toxicity evaluation showed that while benznidazole group caused liver toxicity, the nanoparticles were safe, indicating that this formulation is promising for future evaluation.

Anti-HER2 immunoliposomes for co-delivery of paclitaxel and rapamycin for breast cancer therapy.

Breast cancer is the second leading cause of cancer deaths among women. Paclitaxel (PTX) is used for its treatment, however non-selectivity, rapid systemic clearance and hypersensitivity to the commercially available formulation are major drawbacks. Rapamycin (RAP), an mTOR inhibitor, acts synergistically with PTX, and thus could be used in combination with it. Drug loading into nanocarriers, particularly liposomes, has proven to enhance efficacy and reduce side-effects of chemotherapeutic drugs. Within this context, the functionalization of liposomes with antibodies for overexpressed receptors on tumor surface is a potential strategy to increase specificity and reduce side-effects. Specifically, active targeting of HER2(+) breast cancer cells can be achieved by immunoliposomes consisting of liposomes coated with an anti-HER2 monoclonal antibody, Trastuzumab. Herein, we have synthesized PTX/RAP co-loaded immunoliposomes coated with Trastuzumab, performed physicochemical characterization, and evaluated the formulations for cytotoxicity and uptake in 4T1 (triple negative) and SKBR3 (HER2 positive) cell lines. Furthermore, we aimed to compare the immunoliposomes with liposomes and solution of PTX/RAP in vivo, employing human xenograft HER2-overexpressing tumors in mouse model. The co-loaded immunoliposomes had a mean particle size of 140.3nm, a zeta potential of -9.85mV and drug encapsulation efficiency of 55.87 and 69.51, respectively for PTX and RAP. The functionalization efficiency of Trastuzumab was higher than 70% and the antibody retained HER2 binding activity. Cell studies showed increased cytotoxicity of PTX/RAP for the immunoliposome, compared to the control liposomes in SKBR3 cells, which could be attributed to enhanced uptake mediated through HER2 binding. Furthermore, immunoliposomes were better able to control tumor growth in vivo, with tumor volume averages corresponding to 25.27, 44.38 and 47.78% of tumor volumes of untreated control, PTX/RAP solution and control liposomes, respectively. Taken together, our results support the clinical development of immunoliposomes for targeted delivery of PTX and RAP to HER2-positive breast cancer.

Development and Evaluation of a Nanoemulsion Containing Ursolic Acid: a Promising Trypanocidal Agent : Nanoemulsion with Ursolic Acid Against T. cruzi.

Over a hundred years after the discovery of Chagas disease, this ailment continues to affect thousands of people. For more than 40 years, only two drugs have been available to treat it. Ursolic acid is a naturally occurring terpene that has shown a good trypanocidal action. However, the hydrophobicity of this compound presents a challenge for the development of proper delivery systems. Nanostructured systems are a prominent in delivering lipophilic drugs. Thus, a nanoemulsion containing ursolic acid was developed and had its trypanocidal activity and cytotoxicity evaluated. Pseudo-ternary phase diagrams and hydrophilic-lipophilic balance (HLB) system were used in the development. The system was stable throughout 90 days of testing, as evidenced by turbidimetry analysis and measurements of the droplet size (57.3 nm) and polydispersity index (0.24). Fourier transform infrared spectroscopy and mass spectrometry evidenced drug's integrity in the formulation. An in vitro dissolution profile showed 75% of ursolic acid release after 5 min from the nanoemulsion into the alkaline dissolution medium, while only 20% could be released from a physical mixture after 2 h. Trypanocidal activity and cytotoxicity were evaluated on the CL Brener strain and LLC-MK2 (monkey kidney) fibroblast by chlorophenol red-ß-D-galactoside (CPRG) method. Biological studies showed that the developed formulation was nontoxic and effective against replicant forms of the parasite. A stable and efficient nanoemulsion could be developed to improve the delivery of a promising drug to treat a threatening illness such as Chagas disease.

Rapamycin-loaded Immunoliposomes Functionalized with Trastuzumab: A Strategy to Enhance Cytotoxicity to HER2-positive Breast Cancer Cells.

BACKGROUND: Liposomes have been employed to improve pharmacokinetics and reduce side effects of drugs. They can be functionalized with antibodies for targeted delivery. While the monoclonal antibody trastuzumab has been employed in the therapy of HER2-positive breast cancer, the resistance developed during treatment has been reported. Rapamycin could be used in combination with trastuzumab for improved therapeutic response. OBJECTIVE: In this study, we aimed to develop rapamycin-loaded liposomes and immunoliposomes with trastuzumab, characterize them and evaluate their in vitro cytotoxicity. METHOD: Formulations were prepared by the thin film hydration method and immunoliposome was conjugated to antibody by covalent bond. Characterization involved particle size, polydispersity, zeta potential, encapsulation efficiency, functionalization efficiency, DSC and FTIR assays. Cell studies were conducted through the MTT assay. RESULTS: SPC:Chol:DSPE-PEG formulation prepared at 1:10 drug to lipid ratio presented high encapsulation efficiency, appropriate particle size, low polydispersity, negative zeta potential and colloidal stability. Rapamycin exhibited intermolecular interactions with lipids and underwent crystallinity reduction. Rapamycin-loaded immunoliposomes were prepared with high trastuzumab functionalization efficiency and antibody stability. Cytotoxicity studies showed that the HER2-positive SK-BR-3 cell line was sensitive to trastuzumab, either as free drug or in the context of immunoliposomes, and is more sensitive to rapamycin than the triple negative MDA-MB-231 cells. For MDA-MB-231, the liposomal rapamycin was more cytotoxic than the free drug. Furthermore, the immunoliposomes showed potent cytotoxicity against SK-BR-3 cells. Finally, rapamycin and trastuzumab exhibited in vitro synergistic effect, particularly through immunoliposomes. CONCLUSION: The formulation developed herein has potential for in vivo evaluation.

Co-loaded paclitaxel/rapamycin liposomes: Development, characterization and in vitro and in vivo evaluation for breast cancer therapy.

Paclitaxel and rapamycin have been reported to act synergistically to treat breast cancer. Albeit paclitaxel is available for breast cancer treatment, the most commonly used formulation in the clinic presents side effects, limiting its use. Furthermore, both drugs present pharmacokinetics drawbacks limiting their in vivo efficacy and clinic combination. As an alternative, drug delivery systems, particularly liposomes, emerge as an option for drug combination, able to simultaneously deliver co-loaded drugs with improved therapeutic index. Therefore, the purpose of this study is to develop and characterize a co-loaded paclitaxel and rapamycin liposome and evaluate it for breast cancer efficacy both in vitro and in vivo. Results showed that a SPC/Chol/DSPE-PEG (2000) liposome was able to co-encapsulate paclitaxel and rapamycin with suitable encapsulation efficiency values, nanometric particle size, low polydispersity and neutral zeta potential. Taken together, FTIR and thermal analysis evidenced drug conversion to the more bioavailable molecular and amorphous forms, respectively, for paclitaxel and rapamycin. The pegylated liposome exhibited excellent colloidal stability and was able to retain drugs encapsulated, which were released in a slow and sustained fashion. Liposomes were more cytotoxic to 4T1 breast cancer cell line than the free drugs and drugs acted synergistically, particularly when co-loaded. Finally, in vivo therapeutic evaluation carried out in 4T1-tumor-bearing mice confirmed the in vitro results. The co-loaded paclitaxel/rapamycin pegylated liposome better controlled tumor growth compared to the solution. Therefore, we expect that the formulation developed herein might be a contribution for future studies focusing on the clinical combination of paclitaxel and rapamycin.

Preparation, characterization and evaluation of the in vivo trypanocidal activity of ursolic acid-loaded solid dispersion with poloxamer 407 and sodium caprate

Ursolic acid is a promising candidate for treatment of Chagas disease; however it has low aqueous solubility and intestinal absorption, which are both limiting factors for bioavailability. Among the strategies to enhance the solubility and dissolution of lipophilic drugs, solid dispersions are growing in popularity. In this study, we employed a mixture of the surfactants poloxamer 407 with sodium caprate to produce a solid dispersion containing ursolic acid aimed at enhancing both drug dissolution and in vivo trypanocidal activity. Compared to the physical mixture, the solid dispersion presented higher bulk density and smaller particle size. Fourier Transform Infrared Spectroscopy results showed hydrogen bonding intermolecular interactions between drug and poloxamer 407. X-ray diffractometry experiments revealed the conversion of the drug from its crystalline form to a more soluble amorphous structure. Consequently, the solubility of ursolic acid in the solid dispersion was increased and the drug dissolved in a fast and complete manner. Taken together with the oral absorption-enhancing property of sodium caprate, these results explained the increase of the in vivo trypanocidal activity of ursolic acid in solid dispersion, which also proved to be safe by cytotoxicity evaluation using the LLC-MK2 cell line.

O ácido ursólico é um candidato promissor para o tratamento da doença de Chagas, contudo este fármaco possui baixa solubilidade aquosa e limitada absorção intestinal, ambos os fatores limitantes da biodisponibilidade. Entre as estratégias para potencializar a solubilidade e a dissolução de fármacos lipofílicos, as dispersões sólidas estão crescendo em popularidade. Neste estudo, empregamos mistura dos tensoativos, poloxamer 407 e caprato de sódio, para produzir dispersão sólida contendo ácido ursólico, com o objetivo de aumentar tanto a dissolução do fármaco quanto a atividade tripanocida in vivo. Comparada à mistura física, a dispersão sólida apresentou maior densidade e menor tamanho de partícula. Os resultados da análise de espectroscopia no infravermelho com transformada de Fourier mostraram interações intermoleculares do tipo ligações de hidrogênio entre o fármaco e o poloxamer 407. Os experimentos de difratometria de raio-X revelaram a conversão do fármaco de sua forma cristalina para a forma amorfa, mais solúvel. Consequentemente, a solubilidade do ácido ursólico em dispersão sólida foi aumentada e o fármaco dissolveu-se de maneira mais rápida e completa. Em conjunto com as propriedades promotoras de absorção oral do caprato de sódio, estes resultados explicaram o aumento da atividade tripanocida in vivo do ácido ursólico em dispersão sólida, que também se provou segura após avaliação de citotoxicidade empregando a linhagem celular LLC-MK2.

Liposomes as carriers of hydrophilic small molecule drugs: strategies to enhance encapsulation and delivery.

Although hydrophilic small molecule drugs are widely used in the clinic, their rapid clearance, suboptimal biodistribution, low intracellular absorption and toxicity can limit their therapeutic efficacy. These drawbacks can potentially be overcome by loading the drug into delivery systems, particularly liposomes; however, low encapsulation efficiency usually results. Many strategies are available to improve both the drug encapsulation efficiency and delivery to the target site to reduce side effects. For encapsulation, passive and active strategies are available. Passive strategies encompass the proper selection of the composition of the formulation, zeta potential, particle size and preparation method. Moreover, many weak acids and bases, such as doxorubicin, can be actively loaded with high efficiency. It is highly desirable that once the drug is encapsulated, it should be released preferentially at the target site, resulting in an optimal therapeutic effect devoid of side effects. For this purpose, targeted and triggered delivery approaches are available. The rapidly increasing knowledge of the many overexpressed biochemical makers in pathological sites, reviewed herein, has enabled the development of liposomes decorated with ligands for cell-surface receptors and active delivery. Furthermore, many liposomal formulations have been designed to actively release their content in response to specific stimuli, such as a pH decrease, heat, external alternating magnetic field, ultrasound or light. More than half a century after the discovery of liposomes, some hydrophilic small molecule drugs loaded in liposomes with high encapsulation efficiency are available on the market. However, targeted liposomes or formulations able to deliver the drug after a stimulus are not yet a reality in the clinic and are still awaited.

Solid dispersion of ursolic acid in Gelucire 50/13: a strategy to enhance drug release and trypanocidal activity.

Solid dispersions (SDs) are an approach to increasing the water solubility and bioavailability of lipophilic drugs such as ursolic acid (UA), a triterpenoid with trypanocidal activity. In this work, Gelucire 50/13, a surfactant compound with permeability-enhancing properties, and silicon dioxide, a drying adjuvant, were employed to produce SDs with UA. SDs and physical mixtures (PMs) in different drug/carrier ratios were characterized and compared using differential scanning calorimetry, hot stage microscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), particle size, water solubility values, and dissolution profiles. Moreover, LLC-MK2 fibroblast cytotoxicity and trypanocidal activity evaluation were performed to determine the potential of SD as a strategy to improve UA efficacy against Chagas disease. The results demonstrated the conversion of UA from the crystalline to the amorphous state through XRD. FTIR experiments provided evidence of intermolecular interactions among the drug and carriers through carbonyl peak broadening in the SDs. These findings helped explain the enhancement of water solubility from 75.98 µg/mL in PMs to 293.43 µg/mL in SDs and the faster drug release into aqueous media compared with pure UA or PMs, which was maintained after 6 months at room temperature. Importantly, improved SD dissolution was accompanied by higher UA activity against trypomastigote forms of Trypanosoma cruzi, but not against mammalian fibroblasts, enhancing the potential of UA for Chagas disease treatment.