Josimar O. Eloy

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.

Nanocarriers for Nitric Oxide Delivery

Nitric oxide (NO) is a promising pharmaceutical agent that has vasodilative, antibacterial, and tumoricidal effects. To study the complex and wide-ranging roles of NO and to facilitate its therapeutic use, a great number of synthetic compounds (e.g., nitrosothiols, nitrosohydroxyamines, N-diazeniumdiolates, and nitrosyl metal complexes) have been developed to chemically stabilize and release NO in a controlled manner. Although NO is currently being exploited in many biomedical applications, its use is limited by several factors, including a short half-life, instability during storage, and potential toxicity. Additionally, efficient methods of both localized and systemic in vivo delivery and dose control are needed. One strategy for addressing these limitations and thus increasing the utility of NO donors is based on nanotechnology.

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.

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

&NA; 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 140.3 nm, a zeta potential of −9.85 mV 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. Graphical abstract Figure. No caption available.

Desenvolvimento e validação de um método analítico por CLAE para quantificação de ácido ursólico em dispersões sólidas

Ursolic acid is a natural molecule that presents several pharmacological properties. In this work, an analytical method by RP-HPLC has been developed and validated for quantification of this drug in the solid dispersions, using PEG 6000 and Poloxamer 407 as polymers. The method was specific, linear in the range of 1.0-50.0 µg mL-1 (r<0.99), precise (CV < 5% for both inter- and intra-assays), accurate (maximum deviation of ± 13%), and robust to the parameters evaluated. This method has proved to be simple and useful for ursolic acid determination in solid dispersions, enabling its determination in pharmaceutical dosage form.

Targeted Lipid Nanoparticles for Antisense Oligonucleotide Delivery

Antisense oligonucleotides (AS-ODNs) are short, single-stranded DNA molecules designed to bind specifically to a target messenger RNA (mRNA) and down-regulate gene expression. Despite being a promising class of therapeutics for a variety of diseases, they face major hurdles limiting their clinical application, including low intracellular delivery and poor in vivo stability. Among strategies available to enhance delivery, lipid nanoparticles have gained considerable attention. Active targeting of carriers of AS-ODNs is likely to further enhance delivery efficiency. For that, ligands for overexpressed receptors on the cell surface can be linked to the lipid nanoparticle, facilitating intracellular uptake, resulting in improved efficacy and reduced systemic toxicity. These include cell penetrating peptides (CPPs), transferrin, folate, oligosaccharides, polysaccharides and antibodies. Although targeted-lipid nanoparticles have been shown to enhance intracellular delivery and therapeutic effect of AS-ODNs, no clinical evaluation has been conducted yet. Therefore, more efforts are needed to turn these promising tools into clinical products.

Evaluation of critical parameters for in vitro skin permeation and penetration studies using animal skin models

ABSTRACT In vitro skin permeation/penetration studies may be affected by many sources of variation. Herein, we aimed to investigate the major critical procedures of in vitro skin delivery studies. These experiments were performed with model drugs according to official guidelines. The influence of skin source on penetration studies was studied as well as the use of a cryopreservation agent on skin freezing evaluated by transepidermal water loss, electrical resistance, permeation/penetration profiles and histological changes of the skin. The best condition for tape stripping procedure was validated through the evaluation of the distribution of corneocytes, mass of stratum corneum (SC) removed and amount of protein removed using finger pressure, a 2 kg weight and a roller. The interchangeability of the tape stripping procedures followed by the epidermis and dermis homogenate and the micrometric horizontal cryostat skin sectioning methods were also investigated, besides the effect of different formulations. Noteworthy, different skin sources were able to ensure reliable interchangeability for in vitro permeation studies. Furthermore, an increased penetration was obtained for stored frozen skin compared to fresh skin, even with the addition of a cryoprotectant agent. The best method for tape stripping was the finger pressure followed by the addition of a propylene glycol solvent leading to better SC removal. Finally, no significant difference was found in skin penetration studies performed by different methods suggesting their possible interchangeability. Graphical abstract Figure. No Caption available.

Immunoliposomes: A review on functionalization strategies and targets for drug delivery

Nanoparticles, especially liposomes, have gained prominence in the field of drug delivery for the treatment of human diseases, particularly cancer; they provide several advantages, including controlled drug release, protection of the drug against degradation, improved pharmacokinetics, long circulation, and passive targeting to tumors and inflammatory sites due to the enhanced permeability and retention effect. The functionalization of liposomes with monoclonal antibodies or antibody fragments to generate immunoliposomes has emerged as a promising strategy for targeted delivery to and uptake by cells overexpressing the antigens to these antibodies, with a consequent reduction in side effects. In this review, we address functionalization strategies for the non-covalent and covalent attachment of monoclonal antibodies and their fragments to liposomal surfaces. The main reaction occurs between the sulfhydryl groups of thiolated antibodies and maleimide-containing liposomes. Furthermore, we explore the main targeting possibilities with these ligands for the treatment of a variety of pathologies, including HER2- and EGFR-positive cancers, inflammatory and cardiovascular diseases, infectious diseases, and autoimmune and neurodegenerative diseases, which have not previously been reviewed together. Overall, many studies have shown selective delivery of immunoliposomes to target cells, with promising in vivo results, particularly for cancer treatment. Although clinical trials have been conducted, immunoliposomes have not yet received clinical approval. However, immunoliposomes are promising formulations that are expected to become available for therapeutic use after clinical trials prove their safety and efficacy, and after scaling issues are resolved.

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.

Cetuximab Immunoliposomes Enhance Delivery of 5-FU to Skin Squamous Carcinoma Cells

BACKGROUND Topical chemotherapy of skin cancers is a promising strategy for reduction of side effects and for improvement of patient compliance. The combination of the chemotherapeutic 5-fluouracil (5-FU) and the anti- EGFR antibody cetuximab is a strategy to inhibit tumor growth. Their skin penetration, however, is hampered by their high hydrophilicity, which could be improved by encapsulation in delivery systems. Furthermore, it is a challenge to encapsulate hydrophilic drugs. The conjugation of an antibody to a liposome, maintaining its activity, is also a difficult task. OBJECTIVE Thus, we aimed to develop 5-FU liposomes and cetuximab-conjugated liposomes (immunoliposomes) of 5- FU to improve drug cytotoxicity against skin cancer cells. METHOD We characterized them by particle size, zeta potential, loading efficiency and antibody integrity. To optimize the loading efficiency of 5-FU, a series of liposomes were prepared, using different methods and drug-to-lipid ratios. RESULTS Liposomes containing DSPC and Chol at drug-to-lipid ratio 0.1 prepared by the thin lipid hydration method resulted in the best 5-FU encapsulation and were chosen to conjugate with cetuximab. Cetuximab was directly coupled to preformed liposomes using DSPE-mPEG2000-Mal as an anchor. In A431 skin carcinoma cells, at 72 h, 5-FU liposomes showed a 5-fold lower IC50 than 5-FU solution. Additionally, 5-FU immunoliposomes resulted in a 4-fold lower cetuximab IC50 than cetuximab solution, demonstrating synergism with a combination index lower than 1 and potential to improve 5-FU and cetuximab cytotoxicity. CONCLUSION Liposomes and immunoliposomes containing 5-FU were developed and cetuximab remained active as demonstrated in cell culture studies.