Flávia Sousa

Stability Study Perspective of the Effect of Freeze-Drying Using Cryoprotectants on the Structure of Insulin Loaded into PLGA Nanoparticles

This work aimed to evaluate the influence of a freeze-drying process using different cryoprotectants on the structure of insulin loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles and to assess the stability of these nanoparticles upon 6 months of storage following ICH guidelines. Insulin-loaded PLGA nanoparticles with a size around 450 nm were dehydrated using a standard freeze-drying cycle, using trehalose, glucose, sucrose, fructose, and sorbitol at 10% (w/v) as cryoprotectants. All formulations, except those nonadded of cryoprotectant and added with trehalose, collapsed after freeze-drying. The addition of cryoprotectants increased the nanoparticles stability upon storage. FTIR results showed that insulin maintained its structure after encapsulation in about 88%, decreasing to 71% after freeze-drying. The addition of cryoprotectants prior to freeze-drying increased insulin structural stability an average of up to 79%. Formulations collapsed after freeze-drying showed better protein stabilization upon storage, in special sorbitol added formulation, preserving 76, 80, and 78% of insulin structure at 4 °C, 25 °C/60% RH, and 40 °C/75% RH, respectively. Principal component analysis also showed that the sorbitol-added formulation showed the most similar insulin structural modifications among the tested storage conditions. These findings suggested that regarding nanoparticles stability, cryoprotectants are versatile to be used in a standard freeze-drying, however they present different performances on the stabilization of the loaded protein. Thus, on the freeze-drying of the nanoparticles field, this work gives rise to the importance of the process of optimization, searching for a balance between a good obtainable cake with an optimal structural stabilization of the loaded protein.

Oral films as breakthrough tools for oral delivery of proteins/peptides

Therapeutic proteins and peptides demonstrate unique, peerless, pharmacological characteristics such as high specificity to receptors and superior biological mimicking of physiological mechanisms, resulting in a better therapeutic index compared to conventional chemical-derived drugs. However, proteins also present inherent bioavailability limitations. Thus, this paper proposes several effective tools to improve protein/peptide drugs stability, permeability and pharmacokinetics with special emphasis on oral polymeric films as oral delivery platforms. Indeed, oral films present inherent characteristics that can greatly enhance biological performance of proteins and peptides and patient compliance along with other advantages that are critically discussed in this review. A rational choice of excipients addressed in and manufacture processes are also focused. In addition, possible toxicity issues to be overtaken and critical analysis regarding current market tendencies respecting oral films and protein/peptides along with future prospects are disclosed.

A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles

Monoclonal antibodies have deserved a remarkable interest for more than 40 years as a vital tool for the treatment of various diseases. Still, there is a raising interest to develop advanced monoclonal antibody delivery systems able to tailor pharmacokinetics. Bevacizumab is a humanized immunoglobulin IgG1 used in antiangiogenic therapies due to its capacity to inhibit the interaction between vascular endothelial growth factor and its receptor. However, bevacizumab-based antiangiogenic therapy is not always effective due to poor treatment compliance associated to multiples administrations and drug resistance. In this work, we show a promising strategy of encapsulating bevacizumab to protect and deliver it, in a controlled manner, increasing the time between administrations and formulation shelf-life. Nanoencapsulation of bevacizumab represents a significant advance for selective antiangiogenic therapies since extracellular, cell surface and intracellular targets can be reached. The present study shows that bevacizumab-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles does not impair its native-like structure after encapsulation and fully retain the bioactivity, making this nanosystem a new paradigm for the improvement of angiogenic therapy.

How to overcome the limitations of current insulin administration with new non-invasive delivery systems.

Non-invasive insulin delivery systems have potential to overcome the most pressing problem regarding effective treatment of diabetic patients: therapy compliance. To overcome this disadvantage, non-invasive routes such as oral, buccal, pulmonary, nasal and transdermal have been proposed. These new routes of insulin administration may help to suppress hypoglycemia episodes and aid to control weight gain and post-meal glucose. Despite all efforts the invasive route remains preferential, since studies on insulin administration by non-invasive routes conducted to date have not demonstrated clinical efficacy and safety, including some products introduced in the market. Therefore, the aim of this review is to make an update of the current state of administration of insulin by non-invasive routes as alternatives to the conventional invasive route.

Nanoparticles for the delivery of therapeutic antibodies: Dogma or promising strategy?

ABSTRACT Introduction: Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy. Another disadvantage inherent to antibody-based therapy is the limited stability of antibodies and the low level of tissue penetration. The use of nanoparticles as delivery systems for antibodies allows for a reduction in antibody dosing and may represent a suitable alternative to increase antibody stability Areas covered: We discuss different nanocarriers intended for the delivery of antibodies as well as the corresponding encapsulation methods. Recent developments in antibody nanoencapsulation, particularly the possible toxicity issues that may arise from entrapment of antibodies into nanocarriers, are also assessed. In addition, this review will discuss the alterations in antibody structure and bioactivity that occur with nanoencapsulation. Expert opinion: Nanocarriers can protect antibodies from degradation, ensuring superior bioavailability. Encapsulation of therapeutic antibodies may offer some advantages, including potential targeting, reduced immunogenicity and controlled release. Furthermore, antibody nanoencapsulation may aid in the incorporation of the antibodies into the cells, if intracellular components (e.g. intracellular enzymes, oncogenic proteins, transcription factors) are to be targeted.

Functionalizing PLGA and PLGA Derivatives for Drug Delivery and Tissue Regeneration Applications

Poly(lactic-co-glycolic) acid (PLGA) is one of the most versatile biomedical polymers, already approved by regulatory authorities to be used in human research and clinics. Due to its valuable characteristics, PLGA can be tailored to acquire desirable features for control bioactive payload or scaffold matrix. Moreover, its chemical modification with other polymers or bioconjugation with molecules may render PLGA with functional properties that make it the Holy Grail among the synthetic polymers to be applied in the biomedical field. In this review, the physical-chemical properties of PLGA, its synthesis, degradation, and conjugation with other polymers or molecules are revised in detail, as well as its applications in drug delivery and regeneration fields. A particular focus is given to successful examples of products already on the market or at the late stages of trials, reinforcing the potential of this polymer in the biomedical field.

Development and validation of a rapid reversed-phase HPLC method for the quantification of monoclonal antibody bevacizumab from polyester-based nanoparticles

Graphical abstract Figure. No caption available. HighlightsBevacizumab is an important human monoclonal antibody approved for the treatment of several cancers.A rapid and excellent alternative quantification method for bevacizumab within nanoparticles.A rapid and excellent RP‐HPLC with fluorescence detection method for bevacizumab quantification from nanoparticles.This method allows the characterization of nanoparticulate systems with bevacizumab encapsulated. Abstract Bevacizumab is a powerful human monoclonal antibody approved worldwide for treatment of several types of cancer and ocular diseases due to its potential as antiangiogenic drug. Nowadays, in order to improve the monoclonal antibody‐based therapy, attempts have been focused in the formulation of these biomacromolecules into nanoparticles. Thus, the aim of this work was to develop and validate a reversed‐phase high‐performance liquid chromatography with fluorescence detection method for the determination of bevacizumab from nanoparticulate systems, according to the International Conference on Harmonization guidelines. Chromatographic analysis were performed on a RP‐C8 column with a mobile phase composed by water‐0.1% (v/v) TFA and acetonitrile‐0.1% (v/v) TFA in gradient mode at a flow rate of 1 mL min−1. Results showed that the proposed method is specific, linear in the range of 10–100 &mgr;g mL−1 (r2 = 0.9997), accurate (recovery rate 100.50 ± 0.85%), precise at the intraday and inter‐day (relative standard deviation less than 1.79%) and robust. The detection and quantification limits were calculated by specific linear calibration curve with less concentrated standard (range of 1–20 &mgr;g mL−1). The LOD was 2.16 &mgr;g mL−1 and LOQ was 6.55 &mgr;g mL−1. This method was also successfully used, for the first time, to quantify and compare the content of bevacizumab encapsulated into poly(lactic‐co‐glycolic acid)‐based nanoparticles before and after lyophilization.

Biophysical study of bevacizumab structure and bioactivity under thermal and pH-stresses

ABSTRACT The evaluation of the structural stability and bioactivity of monoclonal antibodies (mAb) is a crucial step in the development of mAb therapeutic based products, since immunogenicity needs to be avoided. In the present work, a study was carried out to understand the changes on the structure and bioactivity of mAbs induced by different pH and temperature values. Structural changes of bevacizumab were monitored using fluorescence spectroscopy, circular dichroism (CD) and Attenuated total reflectance‐Fourier transform infrared spectroscopy (ATR‐FTIR). The secondary and tertiary structural content was monitored at six different pH values and at room temperature, upon heating up to 85 °C and upon cooling down to 20 °C. Furthermore, the temperature induced conformational changes were continuously monitored from 20 °C to 85 °C using fluorescence spectroscopy and circular dichroism, allowing to monitor the melting temperature of the protein at different pH values. The results showed that the thermal denaturation of bevacizumab was irreversible at all pH value. The conformational changes induced by pH were higher at extreme pH values (5, 9 and 10) than neutral pH. Thermal stability studies showed that pH 6 was the pH that confer bevacizumab the highest structural stability. These studies were confirmed by in vitro studies, where bevacizumabs bioactivity was measured by cell viability/proliferation at all pH values at room temperature, and it was found a higher bioactivity for pH 6. Biophysical and biological studies were correlated in order to understand the importance of the modifications in bevacizumab structural content on its bioactivity. However, a decrease in bevacizumabs bioactivity was observed for pH 8, 9 and 10. Overall, this work demonstrated the usefulness of the spectroscopy techniques for estimating the stability of therapeutic mAb during formulation development. Graphical abstract Figure. No Caption available.

Polyester-Based Nanoparticles for the Encapsulation of Monoclonal Antibodies

Aliphatic polyesters have been widely explored for biomedical applications (e.g., drug delivery systems, biomedical devices, and tissue engineering). Recently, polyesters have been used in nanoparticle formulations for the controlled release of monoclonal antibodies (mAbs) for the enhanced efficacy of antibody-based therapy. Polyester-based nanoparticles for mAb delivery provide decreased antibody dosage, increased antibody stability and protection and longer therapeutic action, ultimately translating to an increased therapeutic index. Additionally, nanoencapsulation holds the potential for the selective cellular recognition and internalization of mAbs, in the disease context when intracellular organelles and molecules (e.g., enzymes, transcription factors and oncogenic proteins) are the preferred target. We present here a detailed method to prepare mAb-loaded polyester-based nanoparticles and the various techniques to characterize the resulting nanoparticles and mAb structure. Finally, we highlight different biological approaches to assess the in vitro bioactivity of the antibody upon nanoparticle release.

Cell-based in vitro models for nasal permeability studies

Abstract Nasal drug delivery has been highly investigated by the scientific community as a noninvasive alternative route for the administration of drugs for both topical and systemic action. Authorization of nasal drug marketing requires testing of the best formulation, pharmacokinetics, and pharmacodynamics of drugs along with mechanistic aspects of nasal absorption. Nasal permeability may be assessed as recurring to defined and controlled in vitro studies. Thus, primary cell cultures of human nasal epithelium and human nasal cell lines may be used as in vitro models to study drug permeation. This chapter describes cell culture techniques for nasal drug permeability, including cell seeding and maintaining techniques along with permeability assays per se.