Biodegradable drug-eluting stents: Targeting urothelial tumors of upper urinary tract

INTRODUCTION & OBJECTIVES: Urothelial tumors of upper urinary tract are ranked among the most common types of cancers worldwide. The current standard therapy to prevent recurrence is intravesical Bacillus Calmetteâ Guerin (BCG) immunotherapy, but it presents several disadvantages such as BCG failure and intolerance. Another way is to use chemotherapy, which is generally better tolerated that BCG. In this case, drugs such as epirubicin, doxorubicin, paclitaxel and gemcitabine are used. Nevertheless, intravesical chemotherapy only prevents recurrence in the short-term. These failings can be partially attributed to the short residence time and low bioavailability of the drug within the upper urinary tract and the cancer cells, resulting in a need for frequent drug instillation. To avoid these problems, biodegradable ureteral stents impregnated by supercritical fluid CO2 (SCF) with each of the four anti-cancer drugs were produced. MATERIAL & METHODS: Four formulations with different concentrations of gelatin and alginate and crosslink agent were tested and bismuth was added to confer radiopaque properties to the stent. The preliminary in vivo validation studies in female domestic pigs was conducted at the University of Minho, Braga, after formal approval by the institutionâ s review board and in accordance with its internal ethical protocol for animal experiments. Paclitaxel, epirubicin, doxorubicin and gemcitabine were impregnated in the stents and the release kinetics was measured in artificial urine solution (AUS) for 9 days by UV spectroscopy in a microplate reader. The anti-tumoral effect of the developed stents in transitional cell carcinoma (TCC) and HUVEC primary cells, used as control, was evaluated. RESULTS: The in vivo validation of this second-generation of ureteral stents performed was herein demonstrated. Biodegradable ureteral stents were placed in the ureters of a female pigs, following the normal surgical procedure. The animals remained asymptomatic, with normal urine flow. The in vitro release study in AUS of the stent impregnated showed a higher release in the first 72h for the four anti-cancer drugs impregnated after this time the plateau was achieved and the stent degraded after 9 days. The direct and indirect contact of the anti-cancer biodegradable stents with the TCC and HUVEC cell lines confirm the anti-tumor effect of the stents impregnated with the four anti-cancer drugs, reducing around 75% of the viability of the TCC cell line after 72h and no killing effect in the HUVEC cells. CONCLUSIONS: The use of biodegradable ureteral stent in urology clinical practice not only reduce the stent-related symptoms but also open new treatment therapyâ s, like in urothelial tumors of upper urinary tract. Furthermore, we have demonstrated the clinical validation in vivo pig model. This study has thus shown the killing efficacy of the anti-cancer drug eluting biodegradable stents in vitro for the TCC cell line, with no toxicity observed in the control, non-cancerous cells.The direct and indirect contact of the anti-cancer biodegradable stents with the TCC and HUVEC cell lines confirm the anti-tumor effect of the stents impregnated with the four anti-cancer drugs, reducing around 75% of the viability of the TCC cell line after 72h and no killing effect in the HUVEC cells. This study has thus shown the killing efficacy of the anti-cancer drug eluting biodegradable stents in vitro for the TCC cell line, with no toxicity observed in the control, non-cancerous cells

Targeting urothelial tumors of upper urinary tract with drug-eluting stents impregnated by supercritical fluids

Urothelial tumors of upper urinary tract are ranked among the most common types of cancers worldwide and it has been considered as one of the more expensive to treat due of its long-term propensity of recurrence. The current standard therapy to prevent recurrence is intravesical Bacillus Calmette–Guerin (BCG) immunotherapy, but it presents several disadvantages such as BCG failure and intolerance. Another way is to use chemotherapy, that has been reported to be generally better tolerated that BCG. In this case, drugs such as epirubicin, doxorubicin, paclitaxel and gemcitabine are used. Nevertheless, intravesical chemotherapy only prevents recurrence in the short-term[1], [2]. These failings can be partially attributed to the short residence time and low penetration of the drug within the upper urinary tract and the cancer cells, resulting in a need for frequent drug instillation [3]. To avoid these problems, biodegradable ureteral stents impregnated by supercritical fluid CO2 (SCF) with each of the four anti-cancer drugs were produced (figure 1). Four types of drug-eluting biodegradable stents were studied, impregnated with paclitaxel, epirubicin, doxorubicin and gemcitabine. The release kinetics of the impregnated drugs from the anti-cancer drug-eluting stents was measured in artificial urine solution (AUS) for 9 days. The in vitro drugs release from the impregnated biodegradable ureteral stents was analyzed using a microplate reader. The in vitro release study in AUS showed a higher release in the first 72h for the four anti-cancer drugs impregnated after this time the plateau was achieved and the stent degrades after 9 days. Regarding the amount of impregnated drugs by SCF the gemcitabine showed higher amount (109 μg) and the lower amount was obtained for paclitaxel (67 ng). The diffusion coefficient and the impregnation yield were calculated. The anti-tumoral effect of the developed stents in transitional cell carcinoma (TCC) - T24 cell lines was evaluated. T24 cell line was exposed to graded concentrations (0.01 to 2000 ng/ml) of the four drugs for both 4 and 72 hours to determine the sensitivities to each drug (IC50). Toxicity as a result of both direct and indirect contact of the cell lines with the different material conditions of biodegradable stent were studied. The four anti-cancer drugs showed a concentrationdependent inhibitory effect on the T24 and HUVEC cell lines with IC50’s for paclitaxel of 7.30ng and 501.50ng, respectively. The T24 cell line shows to be more sensitive than HUVEC cell line for all the anti-cancer drugs tested. The direct and indirect contact of the anti-cancer biodegradable stents with the T24 and HUVEC cell lines confirm the anti-tumor effect of the stents impregnated with the four anti-cancer drugs, reducing around 75% of the viability of the T24 cell line after 72h and no killing effect in the HUVEC cells. Finally, this study has shown the killing efficacy of the anti-cancer drug eluting biodegradable stents in vitro for the T24 cell lines, with no toxicity observed in the control, non-cancerous cells.Luso­- American Foundation's Grant for Internships in the University of California, Berkeley, 2015/CON5/CAN8; FCT PhD Grant (SFRH/BD/97203/2013); European Union's Seventh Framework Programme (FP7/2007­2013) under grant agreement n° REGPOT­CT2012­316331­ POLARIS; Project “Novel smart and biomimetic materials for innovative regenerative medicine approaches (Ref.: RL1 ­ ABMR ­ NORTE­01­0124­FEDER­000016)” cofinanced by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF

Shielding AZ91D-1%Ca from corrosion through ultrasound melt treatment: a study for stent design

Magnesium-based materials show great potential for producing biodegradable stents, but their high corrosion rates are a roadblock. This study investigates whether ultrasound melt treatment can change the corrosion response of an extruded AZ91D-1.0%Ca (wt.%) in Earle's Balanced Salt Solution by tailoring the intermetallics' morphology in the as-extruded state. The results showed that the wires from ultrasound-treated ingots corroded faster than non-treated ones in immersion for up to 6 hours. This trend shifted for longer periods, and ultrasound-treated material showed lower corrosion rates and uniform corrosion, while the non-treated material displayed localized corrosion signs. Tensile testing of the wires demonstrated that immersion in EBSS lowered the tensile strength and elongation at fracture due to material degradation, regardless of the processing route. Nonetheless, this decline was sharper in the non-treated material. These findings suggest that ultrasound melt processing can be a promising method for improving the corrosion resistance of magnesium-based materials, paving the way for their use in manufacturing biodegradable stents.This work was supported by Portuguese FCT under the project UIDB/04436/2020, the doctoral grant PD/BD/140094/2018 and SFRH/BD/145285/2019

A fibrin coating method of polypropylene meshes enables the adhesion of menstrual blood-derived mesenchymal stromal cells: a new delivery strategy for stem cell-based therapies

Polypropylene (PP) mesh is well-known as a gold standard of all prosthetic materials of choice for the reinforcement of soft tissues in case of hernia, organ prolapse, and urinary incontinence. The adverse effects that follow surgical mesh implantation remain an unmet medical challenge. Herein, it is outlined a new approach to allow viability and adhesion of human menstrual blood-derived mesenchymal stromal cells (MenSCs) on PP surgical meshes. A multilayered fibrin coating, based on fibrinogen and thrombin from a commercial fibrin sealant, was optimized to guarantee a homogeneous and stratified film on PP mesh. MenSCs were seeded on the optimized fibrin-coated meshes and their adhesion, viability, phenotype, gene expression, and immunomodulatory capacity were fully evaluated. This coating guaranteed MenSC viability, adhesion and did not trigger any change in their stemness and inflammatory profile. Additionally, MenSCs seeded on fibrin-coated meshes significantly decreased CD4+ and CD8+ T cell proliferation, compared to in vitro stimulated lymphocytes (p < 0.0001). Hence, the proposed fibrin coating for PP surgical meshes may allow the local administration of stromal cells and the reduction of the exacerbated inflammatory response following mesh implantation surgery. Reproducible and easy to adapt to other cell types, this method undoubtedly requires a multidisciplinary and translational approach to be improved for future clinical uses.This work was supported by: SANTANDER BANK: “Convenio de colaboración empresarial en actividades de interés general” to F.M.; FUNDAÇÃO PARA A CIÊNCIA E A TECNOLOGIA (FCT): post-doctoral contract CEECIND/01026/2018 to J.M.S.; INSTITUTO DE SALUD CARLOS III (ISCIII): a “PFIS” contract (FI19/00041) to M.Á.P., a “Sara Borrell” grant (CD19/00048) to E.L.; a “Miguel Servet I” grant (MS17/00021), co-funded by the European Social Fund (ESF) “Investing in your future”, and projects CP17/00021 and PI18/0911, co-funded by the European Regional Development Fund (ERDF) “A way to make Europe” to J.G.C.; a “CIBERCV” grant (CB16/11/00494), co-funded by the ERDF to F.M.S.-M; JUNTA DE EXTREMADURA, CONSEJERÍA DE ECONOMÍA, CIENCIA Y AGENDA DIGITAL: project IB20184 (co-funded by ERDF) to E.L. and M.P.; grant GR18199, co-funded by the ERDF, to F.M.S.-M.; contracts TA18054 to I.J. and TA18011 to J.J.L. (cofinanced by FEDER)

Biodegradable urological stent systems based on natural origin polymers

Tese de Doutoramento (Programa Doutoral em Engenharia de Tecidos Medicina Regenerativa e Células Estaminais)Ureteral stents are one of the most commonly used devices in urological practices. Ureteral stents are used for temporary or permanent relief of ureteral obstruction to maintain the flow of urine through the ureter after urological surgical procedures or in case of intrinsic or extrinsic obstruction. Nonetheless, they are related with common problems including encrustation, infection, pain and discomfort. As these problems restrict optimal stent function, including maintenance of suitable urine drainage and decrease of hydronephrosis, new ureteral stent biomaterials and designs are required. In last years, progress has been made in the development of biodegradable ureteral stents (BUS) and drug-eluting ureteral stents. These new technologies may provide ureteral stents with increased biocompatibility, decreased vulnerability to encrustation and improved drugelution features. In the present thesis, it is proposed a BUS based on natural origin polymers (i.e., gelatin, alginate and gellan gum) produced after a combination of template gelation and critical point drying. The proposed biodegradable ureteral stent underwent an improvement in mechanical properties and indwelling time throughout the thesis, by testing different formulations and optimizing process parameters. The BUS developed was first successfully validated in vitro showing a degradation profile which occurs by surface erosion, without any fragmentation, in artificial urine solution. The performance of the BUS developed was also tested in vivo, in a porcine model, supporting the biocompatibility and the homogenous degradation observed in vitro. In vivo testing of the BUS compared with a commercial non-degradable ureteral stent has shown less hydronephrosis and capacity to provide a temporary urine drainage as good as the non-degradable commercial stents. In this thesis, the drug-eluting capacity of the developed BUS was also investigated. BUS was impregnated with anti-inflammatory (ketoprofen) and anti-cancer (paclitaxel, doxorubicin, epirubicin and gemcitabine) compounds by supercritical carbon dioxide impregnation. The ketoprofen-eluting biodegradable ureteral stents developed showed a very promising locally delivery of the active compounds within the 72h, which is the timeframe for the description of antiinflammatory agents after the surgical procedure. In the case of drug-eluting BUS impregnated with anti-cancer drugs, cancer cells, when in contact with this stents, after 72h reduced their viability by 75% Results further demonstrate minimal cytotoxic effect of the stents on non-cancer cells used as control. These novel biodegradable ureteral stents might overcome some of the common problems associated with ureteral stenting and avoid the second surgical procedure for stent removal.Os cateteres ureterais são dos dispositivos mais comumente utilizados em Urologia. Estes dispositivos são utilizados para o alívio temporário ou permanente da obstrução ureteral no sentido de manter o fluxo de urina através do ureter dos rins para a bexiga, após procedimentos cirúrgicos urológicos ou em caso de obstrução intrínseca ou extrínseca. No entanto, estão relacionados com problemas comuns como incrustação, infeção, dor e desconforto. Estes limitam a função do cateter, nomeadamente a manutenção da drenagem urinária e a atenuação da hidronefrose, daí a necessidade de desenvolvimento de novos biomateriais e modelos de cateteres ureterais. Nos últimos anos, observaram-se progressos no âmbito do desenvolvimento de cateteres ureterais biodegradáveis (BUS) e de cateteres ureterais com libertação de fármacos. Estas novas tecnologias levam à produção de cateteres ureterais com uma melhor biocompatibilidade, menor vulnerabilidade à incrustação e possibilidade de libertação localizada de fármacos. Na presente tese, propõe-se um BUS baseado em polímeros de origem natural (i.e., gelatina, alginato e goma de gelano) produzidos por uma combinação de vários passos de processamento e secagem supercrítica. O cateter ureteral biodegradável proposto foi sendo melhorado noque diz respeito as propriedades mecânicas e ao tempo de permanência, testando-se diferentes formulações e otimizando-se diferentes parâmetros do processo. O BUS desenvolvido foi inicialmente validado com sucesso In vitro, mostrando numa solução artificial de urina um perfil de degradação que ocorre por erosão superficial, sem qualquer fragmentação. Posteriormente, o BUS foi testado in vivo, num modelo suíno, mantendo a biocompatibilidade e degradação homogénea observada in vitro. Na comparação in vivo do BUS desenvolvido com um cateter ureteral comercial não degradável, o primeiro mostrou menor hidronefrose e semelhante capacidade de drenagem urinária temporária. Neste trabalho foi também investigada a capacidade de libertação de fármacos. Os BUS foram impregnados com compostos anti-inflamatórios (cetoprofeno) e anticancerígenos (paclitaxel, doxorrubicina, epirubicina e gencitabina) por impregnação com dióxido de carbono supercrítico. Os BUS com cetoprofeno revelaram uma libertação local muito promissora dos compostos ativos dentro das 72h, período de tempo para a prescrição de agentes anti-inflamatórios após cirurgia. No caso dos BUS impregnados com fármacos anticancerígenos, foi verificado que as células cancerígenas, quando em contacto com estes cateteres, reduziram a sua viabilidade em 75% após 72h. Os resultados demonstraram para além disso um efeito citotóxico mínimo dos cateteres impregnados sobre células não cancerígenas utilizadas como controlo. Os resultados obtidos no âmbito desta tese demonstram a possibilidade destes novos cateteres ureterais biodegradáveis desenvolvidos poderem superar muitos dos problemas comuns associados aos cateteres comerciais, evitando assim um segundo procedimento cirúrgico para remoção dos mesmos. para alem disso foi ainda demonstrada a capacidade destes cateteres poderem vir a ser utilizados como agentes de libertação controlada

In vitro and ex-vivo permeability studies of paclitaxel and doxorubicin from drug-eluting biodegradable ureteral stents

A drug-eluting biodegradable ureteral stent (BUS) has been developed as a new approach for the treatment of urothelial tumors of upper urinary tract cancer. In a previous work, this system has proven to be a good carrier for anticancer drugs as a potential effective and sustainable intravesical drug delivery system. BUS has revealed to reduce in 75% the viability of human urothelial cancer cells (T24) after 72 h of contact and demonstrated minimal cytotoxic effect on human umbilical vein endothelial cells (HUVECs) which were used as a control. In this work, we studied the permeability of the anticancer drugs, such as paclitaxel and doxorubicin, alone or released from the BUS developed. We used 3 different membranes to study the permeability: polyethersulfone (PES) membrane, HUVECs cell monolayer, and an ex vivo porcine ureter. The ureter thickness was measured (864.51 mm) and histological analysis was performed to confirm the integrity of urothelium. Permeability profiles were measured during 8 h for paclitaxel and doxorubicin. The drugs per se have shown to have a different profile and as expected, increasing the complexity of the membrane to be permeated, the permeability decreased, with the PES being more permeable and the ex vivo ureter tissue being less permeable. The molecular weight has also shown to influence the permeability of each drug and a higher percentage for doxorubicin (26%) and lower for paclitaxel (18%) was observed across the ex vivo ureter. The permeability (P), diffusion (D), and partition (Kd) coefficients of paclitaxel and doxorubicin through the permeable membranes were calculated. Finally, we showed that paclitaxel and doxorubicin drugs released from the BUS were able to remain in the ex vivo ureter and only a small amount of the drugs can across the different permeable membranes with a permeability of 3% for paclitaxel and 11% for doxorubicin. The estimated amount of paclitaxel that remains in the ex vivo ureter tissue is shown to be effective to affect the cancer cell and not affect the noncancer cells.The research leading to these results has received funding from Urology Research Grant Jaba Recordati 2015, Portuguese Urology Association, ICVS/3B’seAssociate Laboratory Research Grants, and the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012-316331-POLARIS. The project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” (RL1 - ABMR - NORTE-01-0124- FEDER-000016) cofinanced by North Portugal Regional Operational Programme (ON.2 e O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) is also acknowledged. Alexandre Barros acknowledges his FCT PhD grant SFRH/BD/97203/2013. The authors would like to acknowledge Teresa Oliveira for the technical assistance on the histological analysis.info:eu-repo/semantics/publishedVersio

Gelatin-based biodegradable ureteral stents for the treatment of urothelial tumors of the upper urinary tract cancer

Upper urinary tract urothelial carcinoma (UTUC) accounts for 5â 10% of urothelial carcinomas and is a disease that has not been widely studied as carcinoma of the bladder. To avoid the problems of conventional therapies, such as the need for frequent drug instillation due to poor drug retention, we developed a biodegradable ureteral stent (BUS) impregnated by supercritical fluid CO2 (scCO2) with the most commonly used anti-cancer drugs, namely paclitaxel, epirubicin, doxorubicin, and gemcitabine. The release kinetics of anti-cancer therapeutics from drug-eluting stents was measured in artificial urine solution (AUS). The in vitro release showed a faster release in the first 72h for the four anti-cancer drugs, after this time a plateau was achieved and finally the stent degraded after 9 days. Regarding the amount of impregnated drugs by scCO2, gemcitabine showed the highest amount of loading (19.57â μg drug /mg polymer: 2% loaded), while the lowest amount was obtained for paclitaxel (0.067â μg drug /mg polymer: 0.01% loaded). A cancer cell line (T24) was exposed to graded concentrations (0.01 to 2000â ng/ml) of each drug for 4 and 72 hours to determine the sensitivities of the cells to each drug (IC50). The direct and indirect contact study of the anti-cancer biodegradable ureteral stents with the T24 and HUVEC cell lines confirmed the anti-tumoral effect of the BUS impregnated with the four anti-cancer drugs tested, reducing by 75% of the viability of the T24 cell line after 72h and demonstrating minimal cytotoxic effect on HUVECs.info:eu-repo/semantics/publishedVersio

Extraction of collagen/gelatin from the marine demosponge Chondrosia reniformis (Nardo, 1847) using water acidified with carbon dioxide: process optimization

Marine sponges are a rich source of natural bioactive compounds. One of the most abundant valuable products is collagen/gelatin, and therefore, presents an interesting alternative source for pharmaceutical and biomedical applications. We have previously proposed an innovative green technology for the extraction of collagen/gelatin from marine sponges based in water acidified with carbon dioxide. In this work, we have optimized the process operating conditions towards high yields and collagen quality as well as to reduce extraction procedure duration and energy consumption. The process extraction efficiency is higher than 50%, corresponding to a yield of approximately 10% of the sponge dry mass, obtained for mild operating conditions 10 bars and 3h. The extracted material was characterized by Scanning Electron Microscopy (SEM), Rheology, Fourier Transformed Infrared Spectroscopy (FTIR), Circular Dichroism (CD), Aminoacid Analysis and SDS-PAGE. The extracts were found to be composed of highly pure mixtures of collagen and gelatin with similar properties of collagen isolated from other marine sources. The cytotoxicity evaluation, performed with L929 cells, has proven the biocompatibility of the material extracted. Overall, the results obtained demonstrate the efficiency of this approach and the high industrial potential of this technology to obtain marine collagen/gelatin with properties suitable for biomedical applications.FCT through Grant EXP/QEQ-EPS/0745/2012, SWIMS (Subcritical Water Isolation of compounds from Marine Sponges)European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement numbers REGPOT-CT2012-316331-POLARIS and KBBE-2010-266033 (project SPECIAL)European Regional Development Fund (ERDF) under the project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” RL1-ABMR-NORTE-01- 0124-FEDER-000016), cofinanced by North Portugal Regional Operational Programme (ON.2, O Novo Norte), under the National Strategic Reference Framework (NSRF)European Research Council grant agreement ERC-2012- ADG 20120216-321266 for project ComplexiT

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved