Smart carriers and nanohealers:A nanomedical insight on natural polymers

Biodegradable polymers are popularly being used in an increasing number of fields in the past few decades. The popularity and favorability of these materials are due to their remarkable properties, enabling a wide range of applications and market requirements to be met. Polymer biodegradable systems are a promising arena of research for targeted and site-specific controlled drug delivery, for developing artificial limbs, 3D porous scaffolds for cellular regeneration or tissue engineering and biosensing applications. Several natural polymers have been identified, blended, functionalized and applied for designing nanoscaffolds and drug carriers as a prerequisite for enumerable bionano technological applications. Apart from these, natural polymers have been well studied and are widely used in material science and industrial fields. The present review explains the prominent features of commonly used natural polymers (polysaccharides and proteins) in various nanomedical applications and reveals the current status of the polymer research in bionanotechnology and science sectors

Terapeuttiseen tarkoitukseen pohjautuva ihmisen retinan pigmenttiepiteelin solulinjan (ARPE-19) mikrokapselointi alginaattiin ja kylmäkuivaus

Cells have multiple functions in the body, including maintenance of the tissue structure and physiological homeostasis. The cells express and secrete proteins and other factors that exert actions in other cells. These principles form the underlying basis for cell therapy and cell transplantations. Transplanted cells can be used to regenerate tissue structures and homeostasis or they can be used as platform for secretion of therapeutic molecules. Biomaterials can be used to augment the cell growth, differentiation and viability in cell therapy. In addition, the biomaterial matrix may help the surgical placement of the cells into the target site. Importantly, the biomaterial may protect the cell from the immunological and inflammatory reactions after transplantation. The immunological protection of the transplanted therapeutic cells is based to selectively permeable artificial membrane. The membrane prevents the passage of high-molecular weight substances such as large antibodies and cytotoxic immune cells, but permits the passage of smaller molecules, like the secreted therapeutic molecules, nutrients, waste products and oxygen. Lately the interest in cell encapsulation and biomaterial cell interactions has increased due to the emerging techniques of cellular engineering and stem cell differentiation. Storage of microencapsulated cells in freeze-dried form would improve the logistics of the cell therapies (e.g. shipment to the hospitals for reconstitution and use). Otherwise, the microencapsulated cells should be kept viable in continuous culture conditions. The goal of this work was to evaluate alginate based microencapsulation of retinal pigment epithelial cell line (ARPE-19) for cell therapy. Cell viability was evaluated with stably expressed secreted alkaline phosphatase (SEAP), live/dead imaging and oxygen consumption. An empirical kinetic model was built based on FITC-dextran release and protein secretion to describe, release and potential accumulation of therapeutic proteins in the cell microcapsules. Primary animal experiments were done to evaluate the protein release and functionality in the cell microcapsules. Alginate based cell microcapsules were frozen and freeze-dried in order to evaluate the possibility for cell microcapsule preservation in dry powder form. In conclusion, ARPE-19 is a potential cell line for long-term cell therapy based on the expression of transgenes. ARPE-19 cells remain vital in the alginate microcapsules, and they are able to express stably transfected transgene over long periods (at least 20 months). The best cell viability was obtained with alginate microcapsules with calcium and barium cross-linking. This method results in adequate pore sizes that allowed secretion of SEAP. The same microcapsules showed biocompatibility after intraperitoneal administration in preliminary animal experiments. Empirical kinetic simulation model was able to predict the possibility of accumulation inside the alginate microcapsules and demonstrated that the accumulation potential depends on the microcapsule structure. Lyophilization of the cell microcapsules showed that the cells were able to retain some viability during freeze-drying and reconstitution when lyoprotectants were used.Solut ylläpitävät kudosten rakenteita ja fysiologista homeostaasia erittämillään proteiineilla ja muilla tekijöillä. Soluterapia ja solujen transplantaatio pohjautuvat näihin periaatteisiin. Transplantoidut solut voivat uudelleen muodostaa tuhoutuneita kudosrakenteita ja fysiologisen homeostaasin, tai ne voivat tuottaa ja erittää jotakin terapeuttisia molekyylejä. Soluterapiassa biomateriaalit voivat edistää solujen kasvua, erilaistumista ja elävyyttä. Biomateriaalimatriisi voi myös auttaa solusiirteen sijoittumista oikeaan paikkaan. Biomateriaali voi myös suojata transplantoituja soluja immunologisilta ja tulehduksellisilta reaktioilta. Terapeuttisten solujen immunologinen suojaus perustuu selektiivisesti läpäisevään keinotekoiseen kalvoon. Kalvo estää isojen molekyylien kuten vasta-aineiden ja sytotoksisten solujen pääsyn kalvon sisään, mutta se läpäisee pienempiä molekyylejä kuten tuotetun terapeuttisen molekyylin, hapen, ravintoaineet ja solun metaboliatuotteet. Kiinnostus solukapselointiin on lisääntynyt ja poikinut useita solukapselointitekniikoita, sekä tuntemusta solujen ja biomateriaalien interaktioista ja kantasolujen erilaistumisesta. Mikrokapseloitujen solujen terapeuttinen potentiaali olisi paljon suurempi jos ne voitaisiin säilöä kylmäkuivatussa muodossa. Tämä edesauttaisi mm. kuljetusta sairaaloihin sekä säilytystä joka muutoin vaatii soluviljelyolosuhteet. Tämän työn päämääränä oli määrittää alginaattiin mikrokapseloidun retinan pigmenttiepiteelisolu-linjan (ARPE-19) potentiaalisuus terapeuttisessa käytössä. Kapseloitujen solujen elävyyttä tutkittiin eri menetelmillä. Tutkimuksen aikana havaittiin että solujen tuottama proteiini kertyi joihinkin kapseleihin ja kertymistä pyrittiin mallittamaan empiirisellä kineettisellä mallituksella. Malli rakennettiin proteiinin ja fluoresoivien merkkiaineiden vapautumiskokeiden perusteella. Solukapseleilla tehtiin primääriset eläinkokeet kuvaamaan proteiinin vapautumista ja solukapselin toiminnallisuutta elimistössä. Alginaattisolukapseleiden mahdollista säilömistä kuivana jauhemuotona määritettiin jäädyttämällä ja kylmäkuivaamalla solukapselit. Työn johtopäätöksenä on, että ARPE-19 -solulinja on potentiaalinen pitkä-aikaiseen transgeeniseen terapiaan. ARPE-19 -solut säilyivät elinkykyisinä alginaattimikrokapseleissa ja erittivät transgeenistä proteiinia ainakin 20 kuukauden ajan. Paras elävyys todettiin alginaattimikrokapseleilla jotka ristisidottiin kalsiumilla ja bariumilla. Tämä valmistusmenetelmä takasi matriisiin tarpeeksi suuren huokoskoon joka mahdollistaa erittyvän proteiinin vapautumisen. Samat kapselit näyttivät omaavan hyvän bioyhteensopivuuden vatsaonteloon annon jälkeen. Empiirinen kineettinen simulaatiomalli pystyi näyttämään että proteiinin kertyminen riippuu mikrokapselin rakenteesta. Solukapseleiden kylmäkuivaus osoitti että kuivatuissa soluissa säilyi joitakin elintoimintoja ja kapselin rakenne palautui kun se palautettiin nesteympäristöön

Biopolymers – Application in Nanoscience and Nanotechnology

In order to reduce the use of non-renewable resources and to minimize the environmental pollution caused by synthetic materials, the quest for utilizing biomaterials is on a rise. Biopolymers in nature are produced by a range of microorganisms and plants. Biopolymers produced by microorganisms require specific nutrients and controlled environmental conditions. This chapter discusses the recent developments and trends of biopolymers especially in the field of nanotechnology. A basic introduction regarding biopolymers is included at the beginning of the chapter. A detailed discussion on various characterization techniques used for characterizing biopolymers and various frequently used biopolymers is also included. Applications of biopolymers in various fields, especially in the field related to nanoscience and nanotechnology, is elaborated at the end of the chapter. Biopolymers together with nanotechnology have already found many applications in various fields including water treatment, biomedical application, energy sector, and food industry. This chapter is intended to give an overview on the importance of biopolymers in nanotechnology-based applications

Selective functionalization of electrospun fibres

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013A engenharia de tecidos é uma área multidisciplinar da engenharia biomédica que articula conceitos da química, física, engenharia e medicina com o objetivo de recuperar ou substituir uma função perdida de determinado órgão ou tecido. Um dos principais desafios desta área da biotecnologia é a criação de matrizes tridimensionais biocompatíveis e biodegradáveis que sejam capazes de garantir um suporte físico e bioquímico adequado à regeneração celular. Assim, as características mecânicas, químicas e biológicas destas matrizes devem ser adaptadas ao ambiente celular que se pretende reproduzir, dando origem quer à resposta celular específica das células cultivadas na matriz, quer à otimização da resposta fisiológica do próprio organismo. Com efeito, dependendo da função a que se destina, as matrizes usadas em engenharia de tecidos variam tanto no biomaterial que lhes dá origem como na técnica de fabricação utilizada. As vantagens dos polímeros face aos outros materiais tais como biocompatibilidade, biodegradação, alta porosidade e boas propriedades mecânicas, tornam-nos no tipo de material mais utilizado na construção de matrizes tridimensionais. É o caso do copolímero PolyActive, já aprovado pela Food and Drug Administration (FDA) e utilizado em múltiplas aplicações em engenharia de tecidos, com especial destaque para a regeneração óssea. A versatilidade deste polímero está estreitamente relacionada com o rácio dos segmentos químicos que o constituem, um segmento hidrofílico de Politereftalato de etileno (PEOT) e outro hidrofóbico de Poli(tereftalato de butileno) (PBT), que ao ser modificado permite o controlo das propriedades mecânicas e químicas do material. Por outro lado, a eletrofiação é uma técnica de fabricação que tem crescido em termos de popularidade pois permite o fabrico de matrizes fibrosas capazes de simular detalhadamente a topografia das fibras de colagénio que compõem a matriz extracelular natural. Tendo tudo isto em conta, neste estudo foram construídas matrizes tridimensionais de PolyActive por eletrofiação capazes de modular e guiar a resposta celular a partir de recursos topográficos e bioquímicos. A topografia das matrizes foi controlada com a introdução de elétrodos capazes de influenciar o campo elétrico e, assim, alinhar as fibras de PolyActive durante o processo de eletrofiação, que ocorreu num ambiente controlado para garantir a reprodução das propriedades das fibras. Já a incorporação de biomoléculas na superfície das fibras foi conseguida a partir da investigação de duas estratégias distintas. Numa das abordagens, matrizes fibrosas de dois tipos de PolyActive (1000PEOT70PBT30 e 300PEOT55PBT45) foram expostas a irradiação ultravioleta (UV) com o objetivo de introduzir grupos químicos na superfície das fibras capazes de aumentar a adesão de biomoléculas. As diferenças entre superfícies tratadas e não tratadas com UV foram analisadas com recurso às técnicas de espetroscopia de infravermelho médio com transformada de Fourier acoplada ao acessório de reflexão total atenuada (ATR-FTIR) e de fotoeletrões excitados por raios X (XPS). Os resultados mostram que os grupos funcionais resultantes da interação da superfície das fibras com o UV dependem do rácio PEOT/PBT e do conteúdo de Polietilenoglicol (PEG) presente no copolímero. Assim, as fibras de 1000PEOT70PBT30 (PA 1000) apresentaram um grande número de grupos carboxilo e hidroxilo na sua superfície devido à degradação do segmento de PEOT e da sua grande cadeia polimérica de PEG após 40 minutos de exposição à radiação UV. Por sua vez, a matriz fibrosa de 300PEOT55PBT45 (PA 300), quando sujeita ao mesmo período de irradiação UV, originou p-benzoquinonas na superfície das suas fibras devido ao alto teor cristalino da sua estrutura. Em ambos os casos, o tratamento UV aumentou as áreas de adesão das proteínas oriundas do meio de cultura celular e por conseguinte a adesão celular tornou-se também mais eficiente. Porém, a resposta celular é dependente não só das características das matrizes, mas também da linha celular utilizada. Por exemplo, as células Schwann de rato mostraram não só preferência pelas áreas ativadas pelo UV, mas também se mostraram sensíveis a pequenas alterações do alinhamento das fibras resultantes das diferenças entre os dois copolímeros. Foi também utilizada uma máscara de níquel para controlar espacialmente a introdução de novos grupos químicos nas superfícies das matrizes fibrosas de PA 300 e PA 1000. A segunda estratégia apresentada consistiu na eletrofiação de fibras de PA 300 com grupos químicos incorporados para uma funcionalização posterior. Basicamente, uma solução polimérica composta por PA 300 e PEG com determinados grupos funcionais numa proporção 4:1 foi sujeita ao processo de eletrofiação, originando fibras de PA 300 com os grupos funcionais do PEG na sua superfície. Esta abordagem inovadora e inédita possibilitou a seleção dos grupos funcionais localizados na superfície das matrizes fibrosas e consequentemente o controlo do tipo de biomoléculas que vão aderir às fibras. Neste estudo foram utilizados dois tipos de PEG funcionalizado: PEG com terminais alcinos ((bis)PEG-Alkyne), que possibilitam a cicloadição azida-alcino com biomoléculas que tenham a função azida; e PEG com grupos terminais de N-hidroxisuccinimida ((bis)PEG-SVA), que facilitam a ligação com proteínas. As superfícies das matrizes de PA 300 + (bis)PEG-SVA e de PA 300 + (bis)PEG-Alkyne foram analisadas recorrendo às técnicas de ATR-FTIR e XPS. No primeiro caso, os resultados provaram a existência de N-hidroxisuccinimida na superfície das fibras, que depois foi confirmada com recurso a microscopia de fluorescência; relativamente às matrizes de PA 300 + (bis)PEG-Alkyne, apesar das técnicas de espectrometria não produzirem resultados conclusivos, foi possível confirmar a presença de alcinos na superfície das fibras a partir das imagens de microscopia de fluorescência. O sucesso da segunda abordagem permite abrir as portas ao aparecimento de novas metodologias de design e fabricação de matrizes biofuncionais, já que torna possível a simulação e controlo do ambiente bioquímico que influencia as respostas celulares de uma forma simples e eficiente.The principal objective of a new generation of tissue engineering scaffolds is to reproduce the spatial and biochemical microenvironmental characteristics of the natural extracellular matrix (ECM) with the purpose of modulating the cell response and consequently enhance tissue repair. There is an enormous variety of scaffolding approaches that highly depend on the biomaterial selection, on the fabrication technique used and on the specific function of the scaffold. In this study, bioactive electrospun scaffolds made of PolyActive (Poly(ethylene oxide terephthalate) / Poly(buylene terephthalate) (PEOT/PBT)) copolymer, capable of combining a spatially organized structure with bioactive factors, was developed. The design and fabrication strategies used to create the scaffolds allow the tailoring of the scaffold’s function by manipulating the introduction of specific chemical groups on its surface for further selective immobilization of complex biomolecules, resulting in the desired cell response. In one approach, the surface of both 300PEOT55PBT45 (PA 300) and 1000PEOT70PBT30 (PA 1000) electrospun fibres were modified via UV exposure, resulting in the introduction of specific functional groups able to improve the protein adsorption process and consequently increase the available areas for cell attachment. A spatial definition of protein adsorption was accomplished by exposing the fibres via patterned mask. An alternative strategy consisted of electrospinning PA 300 fibres with incorporated chemical groups for later functionalization. Alkyne and NHS-esters functional groups were successfully incorporated on the surface of the electrospun fibres via the introduction of specific PEG linkers ((bis)PEG-alkyne and (bis)PEG-SVA) in the electrospinning blend solution. This innovative methodology can be adopted for multiple tissue engineering applications since specific chemical groups can be introduced onto the surface of electrospun fibres, leading to a meticulous selection of the biochemical elements that will be adsorbed and consequently to a detailed control of the cell behaviour

Biomaterials to suppress cancer stem cells and disrupt their tumoral niche

Lack of improvement in the treatment options of several types of cancer can largely be attributed to the presence of a subpopulation of cancer cells with stem cell signatures and to the tumoral niche that supports and protects these cells. This review analyses the main strategies that specifically modulate or suppress cancer stem cells (CSCs) and the tumoral niche (TN), focusing on the role of biomaterials (i.e. implants, nanomedicines, etc.) in these therapies. In the case of CSCs, we discuss differentiation therapies and the disruption of critical signaling networks. For the TN, we analyze diverse strategies to modulate tumor hypervascularization and hypoxia, tumor extracellular matrix, and the inflammatory and tumor immunosuppressive environment. Due to their capacity to control drug disposition and integrate diverse functionalities, biomaterial- based therapies can provide important benefits in these strategies. We illustrate this by providing case studies where biomaterial-based therapies either show CSC suppression and TN disruption or improved delivery of major modulators of these features. Finally, we discuss the future of these technologies in the framework of these emerging therapeutic conceptsFundación BBVA, Proyectos de Investigación en Biomedicina (2014-PO0110) Ministerio de Economía y Competitividad (SAF2014-58189-R, FEDER Funds)S

Adv Healthc Mater

Stem cells are characterized by a number of useful properties, including their ability to migrate, differentiate, and secrete a variety of therapeutic molecules such as immunomodulatory factors. As such, numerous pre-clinical and clinical studies have utilized stem cell-based therapies and demonstrated their tremendous potential for the treatment of various human diseases and disorders. Recently, efforts have focused on engineering stem cells in order to further enhance their innate abilities as well as to confer them with new functionalities, which can then be used in various biomedical applications. These engineered stem cells can take on a number of forms. For instance, engineered stem cells encompass the genetic modification of stem cells as well as the use of stem cells for gene delivery, nanoparticle loading and delivery, and even small molecule drug delivery. The present Review gives an in-depth account of the current status of engineered stem cells, including potential cell sources, the most common methods used to engineer stem cells, and the utilization of engineered stem cells in various biomedical applications, with a particular focus on tissue regeneration, the treatment of immunodeficiency diseases, and cancer.DP2 OD006462/OD/NIH HHS/United StatesR21 NS085569/NS/NINDS NIH HHS/United States1DP20D006462-01/DP/NCCDPHP CDC HHS/United States1R21NS085569-01/NS/NINDS NIH HHS/United States2018-02-13T00:00:00Z25772134PMC5810416vault:2621

Characterisation of cellular responses and gene delivery capabilities within porous agarose scaffolds

A traumatic osteochondral lesion is a severe type of articulating tissue injury which disrupts and causes damage to both surface cartilage and underlying sub-chondral bone layers. Afflicted patients generally present with early symptoms such as joint discomfort and severe pain which if left untreated can ultimately progress to the pathological disease known as post-traumatic osteoarthritis. At this late stage, the recommended treatment option is total joint replacement surgery; a procedure which is highly invasive and only has a finite functional in-situ lifespan. In physically active younger patients, the latter property is sub-optimal and thus more effective early stage interventions are needed to fully repair the osteochondral tissue unit. Gene activated matrices (GAMs) are considered an exciting tissue engineering treatment solution, whereby single or multiple reservoirs of therapeutic DNA payloads are incorporated specifically within a biomaterial matrix to facilitate high quality bone and cartilage tissue formation. However, a limitation of current gene activated matrices is their limited efficacy in gene delivery and lack of spatial patterning during fabrication and incorporation. As osteochondral tissue is complex with gradient-like features, a GAM containing a gradient payload distribution is hypothesised to be more effective than current unpatterned approaches. To achieve this, a novel agarose gel electrophoresis platform was utilised to fabricate porous agarose scaffolds which contained spatially controlled in-situ deposited DNA payloads. Experimentally, this project for the first time sought to comprehensively evaluate and characterise the above platform in terms of its inherent physical biomaterial properties, DNA payload patterning capabilities (DNA-CaP, DNA-PEI), biomaterial adhesion, cytotoxicity profile and 3D transfection performance. To achieve an acceptable osteochondral-like porous biomaterial GAM, it was found that a combination of freeze-drying and higher inherent agarose hydrogel concentrations (>3wt%) were required. Upon conversion into suitably porous scaffolds, the agarose GAM systems were then shown to be cytocompatible but crucially appeared to be lacking inherent 3D cellular adhesion characteristics. Following this major finding, various surface functionalisation methods (fibronectin, LAP-PEO, polydopamine) were tested for scaffold-wide cell adhesion enhancement. The polydopamine coating method appeared remarkably effective in this regard, enhancing C2C12 and Y201 3D cell-scaffold attachment and proliferation specifically after 7 days of seeding. Histological analysis further showed effective differentiation towards osteogenic and chondrogenic lineages when in the presence of respective differentiation media conditions for 4 weeks. 3D transfection analysis revealed, however, that all agarose-polydopamine GAM scaffolds, developed via the electrophoresis patterning platform, failed to induce any tangible gene expression over a 7 day seeding period. Future experimentation is therefore needed to elucidate the exact failure mechanisms shown in this transfection study, with the aim that corrective measures can be generated which can ultimately produce a transfection capable agarose GAM product. In an additional explorative study, it was also shown that an alternative transfection-capable vector type (CCHLV) could be synthesised from the gene expression cassette region of a plasmid vector. This was significantly aided by the novel use of an ELAN type IIS restriction enzyme synthesis strategy, which could more efficiently produce the final nucleic acid vector product in comparison to current gold standard strategies (ELAN type II). Future experimentation is therefore required to fully characterise these newly developed vectors for its 2D and 3D transfection capabilities. Overall, huge strides were made in the advancement of a completely novel early stage continuous gradient GAM concept, of which ultimately serves to expand not only the knowledge and understanding of applying agarose DNA electrophoretic patterning in the field of GAM system development and manufacture, but furthermore elucidates the substantial limitations this specific concept inherently possesses which may restrict its adoption as a osteochondral defect regeneration device

Biodegradable microparticle for stem cell delivery and differentiation

The formation of three-dimensional (3D) models for tissue engineering purpose provides a more conducive environment to enable complex biological interactions and processes between cells, biomaterials and bioactive molecules. Microparticles (MP) can be used as supporting matrix for 3D construct in cells and a carrier to deliver bioactive agents for cell development and differentiation, particularly for bone tissue engineering. Poly(glycerol adipate) (PGA) is a potential polymer for tissue engineering purposes as it is biodegradable and has biocompatibility with several cells. The aim of this study is to modify PGA polymer for MP with well-defined properties for drug encapsulation and release, promote cell-MP interaction and evaluate the osteogenic differentiation with MP incorporation in mouse embryonic stem (mES) and osteoblast cells. The PGA polymer has been modified by substituting 40% pendant hydroxyl groups onto the polymer backbone with stearoyl (C18) groups to increase encapsulation efficiency of drug within MP. Further modification was tethering one carboxyl terminus in PGA polymer with maleimide-poly(ethylene glycol) (MIHA-PEG-NH2) linker for ligand attachment on the surface of MP. Collagen, as a ligand, was modified by attaching iminothiolane to give a functional thiol group for interaction with maleimide group on the surface of 40%C18-PGA-PEG-MIHA MP. The microparticles were prepared using an emulsification method. Dexamethasone phosphate (DXMP) and simvastatin (SIM) were encapsulated within the MP. The MP-cell aggregate formation was evaluated as well as cell metabolism activity. The effect of polymer modification on drug release from MP was evaluated in the cells by analyzing osteogenic differentiation in cells. The MP prepared from modified PGA polymer exhibited high encapsulation efficiency of SIM in MP. By adjusting the formulation parameters, the release of SIM from MP could be extended to 21 days. The collagen attachment on the surface of 40%C18-PGA-PEG-MIHA MP promoted cell metabolic activity and produced more extensive markers related to osteogenic differentiation

Diverse Applications of Nanomedicine

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic. \ua9 2017 American Chemical Society