The freeze-thaw (F/T) method is commonly employed during the processing and handling of drug substances to enhance their chemical and physical stability and obtain pharmaceutical applications such as hydrogels, emulsions, and nanosystems (e.g., supramolecular complexes of cyclodextrins and liposomes). Using F/T in manufacturing hydrogels successfully prevents the need for toxic cross-linking agents; moreover, their use promotes a concentrated product and better stability in emulsions. However, the use of F/T in these applications is limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading, and drug release capacity), which depend on the optimization of process conditions and the kind and ratio of polymers, temperature, time, and the number of cycles that involve high physical stress that could change properties associated to quality attributes. Therefore, is necessary the optimization of F/T conditions and variables. The current research regarding F/T is focused on enhancing the formulations, the process, and the use of this method in pharmaceutical, clinical, and biological areas. The present review aims to discuss different studies related to the impact and effects of the F/T process on the physical, mechanical, and chemical properties (porosity, swelling capacity) of diverse pharmaceutical applications with an emphasis on their formulation properties, the method and variables used, as well as challenges and opportunities in developing. Finally, we review the experimental approach for choosing the standard variables studied in the F/T method applying the systematic methodology of quality by design.

The blood-brain barrier (BBB) is a sophisticated and very selective dynamic interface composed of endothelial cells expressing enzymes, transport systems, and receptors that regulate the passage of nutrients, ions, oxygen, and other essential molecules to the brain, regulating its homeostasis. Moreover, the BBB performs a vital function in protecting the brain from pathogens and other dangerous agents in the blood circulation. Despite its crucial role, this barrier represents a difficult obstacle for the treatment of brain diseases because many therapeutic agents cannot cross it. Thus, different strategies based on nanoparticles have been explored in recent years. Concerning this, chitosan-decorated nanoparticles have demonstrated enormous potential for drug delivery across the BBB and treatment of Alzheimer’s disease, Parkinson’s disease, gliomas, cerebral ischemia, and schizophrenia. Our main objective was to highlight the high potential of chitosan adsorption to improve the penetrability through the BBB of nanoformulations for diseases of CNS. Therefore, we describe the BBB structure and function, as well as the routes of chitosan for crossing it. Moreover, we define the methods of decoration of nanoparticles with chitosan and provide numerous examples of their potential utilization in a variety of brain diseases. Lastly, we discuss future directions, mentioning the need for extensive characterization of proposed nanoformulations and clinical trials for evaluation of their efficacy.

The search for materials and process parameters capable of generating hydrogels for soft tissue engineering applications, based on an experimental design strategy that allows the evaluation of several factors involved in their development and performance, has greatly increased. Nevertheless, the fabrication technique can influence their mechanical properties, swelling, crystallinity, and even their susceptibility to contamination by microorganisms, compromising their performance within the tissue or organ. This study aimed to evaluate the influence of the freeze/thaw technique on different characteristics of polyvinyl alcohol–xanthan gum hydrogel. Methods: this research analyzed the critical variables of the freeze/thaw process through a systematic study of a 2^k factorial design of experiments, such as the proportion and concentration of polymers, freezing time and temperature, and freeze/thaw cycles. Additionally, physicochemical analysis, susceptibility to bacterial growth, and cell viability tests were included to approximate its cytotoxicity. The optimized hydrogel consisted of polyvinyl alcohol and xanthan gum at a 95 : 5 ratio, polymer mixture concentration of 15%, and 12 h of freezing with three cycles of freeze/thaw. The hydrogel was crystalline, flexible, and resistant, with tensile strengths ranging from 9 to 87 kPa. The hydrogel was appropriate for developing scaffolds for soft tissue engineering such as the cardiac and skeletal muscle, dermis, thyroid, bladder, and spleen. Also, the hydrogel did not expose an in vitro cytotoxic effect, rendering it a candidate for biomedical applications.

Melanin is an insoluble, amorphous polymer that forms planar sheets that aggregate naturally to create colloidal particles with several biological functions. Based on this, here, a preformed recombinant melanin (PRM) was utilized as the polymeric raw material to generate recombinant melanin nanoparticles (RMNPs). These nanoparticles were prepared using bottom-up (nanocrystallization—NC, and double emulsion–solvent evaporation—DE) and top-down (high-pressure homogenization—HP) manufacturing approaches. The particle size, Z-potential, identity, stability, morphology, and solid-state properties were evaluated. RMNP biocompatibility was determined in human embryogenic kidney (HEK293) and human epidermal keratinocyte (HEKn) cell lines. RMNPs prepared by NC reached a particle size of 245.9 ± 31.5 nm and a Z-potential of −20.2 ± 1.56 mV; 253.1 ± 30.6 nm and −39.2 ± 0.56 mV compared to that obtained by DE, as well as RMNPs of 302.2 ± 69.9 nm and −38.6 ± 2.25 mV using HP. Spherical and solid nanostructures in the bottom-up approaches were observed; however, they were an irregular shape with a wide size distribution when the HP method was applied. Infrared (IR) spectra showed no changes in the chemical structure of the melanin after the manufacturing process but did exhibit an amorphous crystal rearrangement according to calorimetric and PXRD analysis. All RMNPs presented long stability in an aqueous suspension and resistance to being sterilized by wet steam and ultraviolet (UV) radiation. Finally, cytotoxicity assays showed that RMNPs are safe up to 100 μg/mL. These findings open new possibilities for obtaining melanin nanoparticles with potential applications in drug delivery, tissue engineering, diagnosis, and sun protection, among others.

A biodegradable polymeric system is proposed for formulating peptides and proteins. The systems were assembled through the adsorption of biodegradable polymeric nanoparticles onto porous, biodegradable microspheres by an adsorption/infiltration process with the use of an immersion method. The peptide drug is not involved in the manufacturing of the nanoparticles or in obtaining the microspheres; thus, contact with the organic solvent, interfaces, and shear forces required for the process are prevented during drug loading. Leuprolide acetate was used as the model peptide, and poly(D,L-lactide-co-glycolide) (PLGA) was used as the biodegradable polymer. Leuprolide was adsorbed onto different amounts of PLGA nanoparticles (25 mg/mL, 50 mg/mL, 75 mg/mL, and 100 mg/mL) in a first stage; then, these were infiltrated into porous PLGA microspheres (100 mg) by dipping the structures into a microsphere suspension. In this way, the leuprolide was adsorbed onto both surfaces (ie, nanoparticles and microspheres). Scanning electron microscopy studies revealed the formation of a nanoparticle film on the porous microsphere surface that becomes more continuous as the amount of infiltrated nanoparticles increases. The adsorption efficiency and release rate are dependent on the amount of adsorbed nanoparticles. As expected, a greater adsorption efficiency (~95%) and a slower release rate were seen (~20% of released leuprolide in 12 hours) when a larger amount of nanoparticles was adsorbed (100 mg/mL of nanoparticles). Leuprolide acetate begins to be released immediately when there are no infiltrated nanoparticles, and 90% of the peptide is released in the first 12 hours. In contrast, the systems assembled in this study released less than 44% of the loaded drug during the same period of time. The observed release profiles denoted a Fickian diffusion that fit Higuchi's model (t^1/2). The manufacturing process presented here may be useful as a potential alternative for formulating injectable depots for sensitive hydrophilic drugs such as peptides and proteins, among others.