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Background: Recent advancements in nanomedicine and nanotechnology have expanded the scope of multifunctional nanostructures, offering innovative solutions for targeted drug delivery and diagnostic agents in oncology and nuclear medicine. Nanoparticles, particularly those derived from natural sources, hold immense potential in overcoming biological barriers to enhance therapeutic efficacy and diagnostic accuracy. Papain, a natural plant protease derived from Carica papaya, emerges as a promising candidate for green nanotechnology-based applications due to its diverse medicinal properties, including anticancer properties. Purpose: This study presents a novel approach in nanomedicine and oncology, exploring the potential of green nanotechnology by developing and evaluating technetium-99m radiolabeled papain nanoparticles (99mTc-P-NPs) for imaging breast tumors. The study aimed to investigate the efficacy and specificity of these nanoparticles in breast cancer models through preclinical in vitro and in vivo assessments. Methods: Papain nanoparticles (P-NPs) were synthesized using a radiation-driven method and underwent thorough characterization, including size, surface morphology, surface charge, and cytotoxicity assessment. Subsequently, P-NPs were radiolabeled with technetium-99m (99mTc), and in vitro and in vivo studies were conducted to evaluate cellular uptake at tumor sites, along with biodistribution, SPECT/CT imaging, autoradiography, and immunohistochemistry assays, using breast cancer models. Results: The synthesized P-NPs exhibited a size mean diameter of 9.3 ± 1.9 nm and a spherical shape. The in vitro cytotoxic activity of native papain and P-NPs showed low cytotoxicity in HUVEC, MDA-MB231, and 4T1 cells. The achieved radiochemical yield was 94.2 ± 3.1% that were sufficiently stable (≥90%) for 6 h. The tumor uptake achieved in the 4T1 model was 2.49 ± 0.32% IA/g at 2 h and 1.51 ± 0.20% IA/g at 6 h. In the spontaneous breast cancer model, 1.19 ± 0.20% IA/g at 2 h and 0.86 ± 0.31% IA/g at 6 h. SPECT/CT imaging has shown substantial tumor uptake of the new nanoradiopharmaceutical and clear tumor visualization. 99mTc-P-NPs exhibited a high affinity to tumoral cells confirmed by ex vivo autoradiography and immunohistochemistry assays. Conclusion: The findings underscore the potential of green nanotechnology-driven papain nanoparticles as promising agents for molecular imaging of breast and other tumors through SPECT/CT imaging. The results represent a substantial step forward in the application of papain nanoparticles as carriers of diagnostic and therapeutic radionuclides to deliver diagnostic/therapeutic payloads site-specifically to tumor sites for the development of a new generation of nanoradiopharmaceuticals.

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Bladder cancer (BC) is the tenth most common type of cancer worldwide, affecting up to four times more men than women. Depending on the stage of the tumor, different therapy protocols are applied. Non-muscle-invasive cancer englobes around 70% of the cases and is usually treated using the transurethral resection of bladder tumor (TURBIT) followed by the instillation of chemotherapy or immunotherapy. However, due to bladder anatomy and physiology, current intravesical therapies present limitations concerning permeation and time of residence. Furthermore, they require several frequent catheter insertions with a reduced interval between doses, which is highly demotivating for the patient. This scenario has encouraged several pieces of research focusing on the development of drug delivery systems (DDS) to improve drug time residence, permeation capacity, and target release. In this review, the current situation of BC is described concerning the disease and available treatments, followed by a report on the main DDS developed in the past few years, focusing on those based on mucoadhesive polymers as a strategy. A brief review of methods to evaluate mucoadhesion properties is also presented; lastly, different polymers suitable for this application are discussed.

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The synthesis and engineering of nanomaterials offer more robust systems for the treatment of cancer, with technologies that combine therapy with imaging diagnostic tools in the so-called nanotheranostics. Among the most studied systems, there are quantum dots, liposomes, polymeric nanoparticles, inorganic nanoparticles, magnetic nanoparticles, dendrimers, and gold nanoparticles. Most of the advantages of nanomaterials over the classic anticancer therapies come from their optimal size, which prevents the elimination by the kidneys and enhances their permeation in the tumor due to the abnormal blood vessels present in cancer tissues. Furthermore, the drug delivery and the contrast efficiency for imaging are enhanced, especially due to the increased surface area and the selective accumulation in the desired tissues. This property leads to the reduced drug dose necessary to exert the desired effect and for a longer action within the tumor. Finally, they are made so that there is no degradation into toxic byproducts and have a lower immune response triggering. In this article, we intend to review and discuss the state-of-the-art regarding the use of nanomaterials as therapeutic and diagnostic tools for lung, breast, and prostate cancer, as they are among the most prevalent worldwide.

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Papain is a therapeutic enzyme with restricted applications due to associated allergenic reactions. Papain nanoparticles have shown to be safe for biomedical use, although a method for proper drug loading and release remains to be developed. Thus, the objective of this work was to develop and assess the stability of papain nanoparticles in a prototype semi-solid formulation suitable for dermatological or topical administrations. Papain nanoparticles of 7.0 ± 0.1 nm were synthesized and loaded into carboxymethylcellulose- and poly(vinyl alcohol)-based gels. The formulations were then assayed for preliminary stability, enzyme activity, cytotoxicity studies, and characterized according to their microstructures and protein distribution. The formulations were suitable for papain nanoparticle loading and provided a stable environment for the nanoparticles. The enzyme distribution along the gel matrix was homogeneous for all the formulations, and the proteolytic activity was preserved after the gel preparation. Both gels presented a slow release of the papain nanoparticles for four days. Cell viability assays revealed no potential cytotoxicity, and the presence of the nanoparticles did not alter the microstructure of the gel. The developed systems presented a potential for biomedical applications, either as drug delivery systems for papain nanoparticles and/or its complexes.

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Hydrogels are materials with wide applications in several fields, including the biomedical and pharmaceutical industries. Their properties such as the capacity of absorbing great amounts of aqueous solutions without losing shape and mechanical properties, as well as loading drugs of different nature, including hydrophobic ones and biomolecules, give an idea of their versatility and promising demand. As they have been explored in a great number of studies for years, many routes of synthesis have been developed, especially for chemical/permanent hydrogels. In the same way, stimuli-responsive hydrogels, also known as intelligent materials, have been explored too, enhancing the regulation of properties such as targeting and drug release. By controlling the particle size, hydrogel on the micro- and nanoscale have been studied likewise and have increased, even more, the possibilities for applications of the so-called XXI century materials. In this paper, we aimed to produce an overview of the recent studies concerning methods of synthesis, biomedical, and pharmaceutical applications of macro-, micro, and nanogels.

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The administration of medicines by the oral route is the most used approach for being very convenient. Although it is the most popular, this route also has absorption, and consequently, bioavailability limitations. In this sense, several pharmacotechnical strategies have been used to improve drug absorption, one of which is the use of permeation promoters. Papain is a very versatile plant enzyme that can be used as a permeation promoter of various active compounds. This study aimed to evaluate the safety of papain and the formulation of native papain minitablets to promote in vitro permeation of furosemide through an innovative biomimetic triple co-culture model of Caco-2, HT29-MTX, and Raji cells. Regarding permeation, furosemide and metaprolol concentrations are determined with HPLC; those are used to calculate Papp. Monolayer integrity was evaluated using TEER and Lucifer Yellow. In the presence of papain, TEER decreased two-fold and the Papp of furosemide increased six-fold. The results suggest that native papain minitablets can be used as therapeutic adjuvants to enhance the permeation of drugs significantly improving bioavailability.

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ß-alanine is the rate-limiting point for the endogenous synthesis of carnosine in skeletal muscle. Carnosine has a wide range of implications for health, normal function and exercise performance. Whilst the physiological relevance of carnosine to different tissues remains enigmatic, ß-alanine administration is a useful strategy to investigate the physiological roles of carnosine in humans. Intravenous administration of ß-alanine is an interesting approach to study carnosine metabolism. However, sterilisation is mandatory due to the nature of the administration route. We evaluated whether sterilising doses of gamma radiation damages the molecular structure and leads to the loss of functional characteristics of ß-alanine. Pure ß-alanine was sterilised by gamma radiation in sealed glass vials using a 60Co multipurpose irradiator at a dose rate of 8.5 kGy.hour-1 totalising 10, 20, 25 30 and 40 kGy. The molecular integrity was assessed by X-ray Diffraction and changes in content were determined by High Performance Liquid Chromatography (UV-HPLC) and Triple Quadrupole Mass Spectrometer (HPLC/MS-MS). Sterility assurance was evaluated by inoculation assay. To examine whether functional properties were preserved, ß-alanine was infused in one participant, who rated the level of paraesthesia on the skin using a 0-3 scale. Urinary ß-alanine was quantified before and 24-h following ß-alanine infusion using HPLC-ESI+-MS/MS. Irradiation resulted in no change in the crystal structure of ß-alanine, no degradation, and no new peaks were identified in the dose range assayed. The inoculation assay showed the absence of viable microorganisms in all ß-alanine samples, including those that did not undergo irradiation. Intravenous infusion of ß-alanine resulted in paraesthesia and it detected in the urine as per normal. We conclude that gamma radiation is a suitable technique for the sterilisation of ß-alanine. It does not lead to degradation, damage to the ß-alanine structure, content or loss of function within the evaluated irradiation conditions.

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Safety and biocompatibility assessment of biomaterials are themes of constant concern as advanced materials enter the market as well as products manufactured by new techniques emerge. Within this context, this review provides an up-to-date approach on current methods for the characterization and safety assessment of biomaterials and biomedical devices from a physical-chemical to a biological perspective, including a description of the alternative methods in accordance with current and established international standards.

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Gamma radiation has been shown particularly useful for the functionalization of surfaces with stimuli-responsive polymers. This method involves the formation of active sites (free radicals) onto the polymeric backbone as a result of the high-energy radiation exposition over the polymeric material. Thus, a microenvironment suitable for the reaction among monomer and/or polymer and the active sites is formed and then leading to propagation to form side-chain grafts. The modification of polymers using high-energy irradiation can be performed by the following methods: direct or simultaneous, pre-irradiation oxidative, and pre-irradiation. The most frequently used ones correspond to the pre-irradiation oxidative method as well as the direct one. Radiation-grafting has many advantages over other conventional methods because it does not require the use of catalyst nor additives to initiate the reaction and usually no changes on the mechanical properties with respect to the pristine polymeric matrix are observed. This chapter is focused on the synthesis of smart polymers and coatings obtained by the use of gamma radiation. In addition, the diverse applications of these materials in the biomedical area are also reported, with focus in drug delivery, sutures, and biosensors.

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Polypropylene films were grafted with thermo-responsive N-vinylcaprolactam and pH-responsive N-vinylimidazole polymers by means of gamma radiation using pre-irradiation and direct methods, in order to functionalize the films with thermo- and/or pH-responsiveness. The dependence of grafting yield on parameters such as co-monomer concentration, pre-irradiation dose, temperature, and reaction time was evaluated. The samples were characterized by Fourier transform infrared and X-ray photoelectron spectroscopies, differential scanning calorimetry, thermogravimetric analysis, swelling studies in different solvents, and water contact angle. The grafted copolymers presented thermo- and pH-sensitiveness, highlighting their potential as advanced biomaterials, capable of providing adequate environment for hosting and sustained release of antimicrobial drugs bearing cationic moieties, such as groups of diclofenac, while still exhibiting good cytocompatibility.

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The performance of the standardized extrusion-spheronization technique, operational conditions, formulation parameters and storage of the final product over the bioactivity of papain containing pellets has been evaluated to obtain an insight into the potential of the technique for the manufacture of solid protein formulations. The pellets produced were assayed in terms of biological activity - monitored at each operational stage using N-benzoyl-dl-arginine ρ-nitroanilide as a substrate, and according to the physical properties - evaluated by means of size distribution, apparent density and friability. The produced pellets presented adequate physical and mechanical properties. Monitoring biological activity at each production stage revealed that the most critical steps corresponded to drying and storage, with bioactivity decay ranging from 5 to 30% and 5 to 20% for each process. Dry mixing and extrusion did not hold any influence over papain activity, while wet massing decreased the bioactivity by approximately 0-5% and the spheronization 0-2%. The results varied as a function of the experimental conditions and formulation components. In conclusion, the extrusion--spheronization technique was suitable to produce solid multiparticulate dosage forms for papain, considering the possibility to originate pellets with relatively low bioactivity decay. However, weak points of the technique corresponded to the wet massing and drying stages as well as storage.

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Papain is a proteolytic enzyme with restricted applications due to its limited stability. Cyclodextrins are widely used in pharmaceutical formulations once they are able to form complexes with other molecules, improving their stability and bioavailability. The purpose of the present paper was to analyze complexes formed by papain/hydroxypropyl-beta-cyclodextrin and papain/beta-cyclodextrin by thermal analysis and cytotoxicity tests to verify their possible interactions and toxicological behavior. Complex solutions were prepared at different molar ratios and collected as a function of time to be lyophilized and analyzed. Results showed changes in endothermic events and cytotoxicity profiles. A complex formation for both complexes is observed; nevertheless, beta-cyclodextrin was able to change the enzyme characteristics more efficiently.