Abstract: Controlled delivery of gene transfer vectors is a powerful strategy to enhance the temporal and spatial presentation of therapeutic agents in a defined target. Hydrogels are adapted biomaterials for genedelivery capable of acting as a localized depot of genes while maintaining the long term local availability of DNA vectors at a specific location. Supramolecular hydrogels based on cyclodextrins (CDs) have attracted considerable attention as potential biomaterials in a broad range of drugdelivery applications. Their unique characteristics of thixotropicity and low cytotoxicity due to their production under mild conditions make them potential candidates to form injectable delivery systems. This work aims to provide an overview of the use of CD-based polypseudorotaxane hydrogels as controlled genedelivery systems for different applications in regenerative medicine.
Finally, PCL nanoparticles have also been used for genedelivery in cancer treatment. As an example, specific small interfering RNA (siRNAs) that target the estrogen receptor alpha (ER α ) were encapsulated in PEGylated poly( ε -caprolactone-malic acid) (PEG-PCL/MA) nanocapsules (diameter: 100 – 200 nm, loading efficiency ≈ 70 %), as a novel strategy in the treatment of hormone-dependent breast cancers. Fluorescence quenching assays confirmed the incorporation of siRNA into the nanocapsule core. A persistent loss of ER α (90 % over 5 days) was observed in MCF-7 human breast cancer cells that were treated with this formulation. Furthermore, the intravenous injection of these nanocapsules into estradiol- stimulated MCF-7 cell xenografts led to a significant decrease in tumor growth and a decrease in ER α expression in tumor cells 58 . In this way, several studies have pointed out the benefits of the combination of nanotherapeutic strategies including both gene silencing anddrugdelivery, especially in the treatment of refractory tumors. As an example, PEO-PCL nanoparticles were formulated to efficiently encapsulate MDR-1 silencing siRNA and PTX. Upon administration in MDR SKOV3 TR human ovarian adenocarcinoma cells, siRNA-
Silk-like proteins (SLPs) are another type of recombinant material with demonstrated success in biomedical applications [124, 125]. These biopolymers are designed taking into account the repetitive peptide sequences found in silkworm and spider silk. The most common of all silk variants is probably the hexapeptide GAGAGS from Bombyx mori fibroin, although recombinant spider silk has also been used in nanoparticles for geneanddrugdelivery [126, 127]. In aqueous solution, these silk-derived polymers undergo an essentially irreversible conformation transition from random coil to beta sheet and a subsequent beta sheet aggregation growth accelerated by an increase in temperature. The good biocompatibility and biodegradability shown by these materials, and particularly the mechanical strength of the resulting aggregation products, stimulated the design of chimeric materials such as silk-elastin like recombinamers (SELR). There are many examples of the use of SELRs in drugdeliveryand these have been extensively reviewed in the last few years [9, 128, 129]. However, very few studies reporting the synthesis of SELR-derived nanoparticles have been published [130-133]. These studies demonstrated that the self-assembly of SELR into spherical nanoparticles is a process in which the length of the silk block determines both the kinetics and the size of the aggregates. Thus, an initial temperature-driven aggregation mediated by the elastin block forms nanoparticles (around 40 nm in diameter) that then self-assemble into a nanofibrillar morphology in an annealing time-dependent manner (Figure 5). This coordinated and concomitant dual-gelation mechanism leads to the final maturation into a resistant hydrogel made of the fibrous structures when the concentration of the material and annealing time are appropriate [131, 133]. Consequently, the use of SELRs in nanocarrier synthesis for drugdelivery has excellent potential but requires more in-depth studies of the production procedures and choice of the final target.
A main strategy of regenerative medicine is the construction of a biocompatible scaffold that, in combination with living cells and/or bioactive molecules, replaces, regenerates or repairs damaged cells or tissue. Numerous studies, using nanostructured materials, have demonstrated the validity of this approach for a variety of cell types and the application in regenerating different tissues (such as cardiac, bone, cartilage, skin, bladder, nervous and vascular) by enhancing the biological properties of the cells such as cell adhesion, cell mobility and cell differentiation [3,39–42]. In the in vivo condition the cells are located in three-dimensional (3D) microenvironments, where they are surrounded by other cells and by the extracellular matrix (ECM), whose components, such as collagen, elastin, and laminin, are organized in nanostructures (i.e., fibers, triple helixes, etc.) with specific bioactive motifs that regulate the cell homeostasis. It is therefore essential to develop scaffolds that create synthetic microenvironments providing 3D support, so as to control and direct the cellular behavior and to promote specific cell interactions [43,44]. The goal of nanomaterial-directed stem cell culture is to mimic the properties, both physical and biochemical, of the physiological stem cell niche. Electrospinning is a widely used technique for the production of nanofibers that offers great flexibility in terms of the choice of scaffold material, as well as finer control over the scaffold geometry [45–47]. In the electrospinning process, nanofibres are created as polymeric jets from the surface of a polymeric solution in a high intensity electrostatic field when the electric field overcomes the polymer surface tension. Modulation of spinning parameters such as flow rate and collecting distance, and polymer solution properties such as solvent, concentration, conductivity, and surface tension, properties of the resultant nanofibrous meshes, such as fibre diameter, porosity, mechanical properties and surface topography can be easily controlled [48,49]. Moreover, additional functionalities can be incorporated via protein coatings, or chemical conjugation of specific signalling molecules with great utility in SCs-based therapy . The development of these nanoscaffold-based therapy methods in combination with SCs is an important tool for tissue engineering and regenerative medicine.
SUMMARY. Transdermal drugdelivery systems of venlafaxine hydrochloride were prepared by using combination of hydrophilic (HPMC E15) and hydrophobic (ERS100 and ERL 100) polymers in 1:5, 2:4, 3:3, 4:2, 5:1 ratios by solvent casting technique with 15 % v/w propylene glycol as plasticizer. The drug permeation studies revealed that drug permeation increased proportionally with increasing HPMC ratio where ERS 100 as hydrophobic polymer but in case of ERL 100 as hydrophobic polymer proportional in- crease was not obtained this may be due to increased diffusion path length. The drug permeation kinetics followed zero order profile with diffusion mechanism. The average steady state flux obtained with HPMC: ERL 100 (3:3) was 193.2 μg/cm 2 /h and the same was increased to 257 μg/cm 2 /h with the incorporation of 5
Finally, the quantiﬁcation of transfection by the poly- plexes was necessary for the direct application of these ELRs in gene therapy (Fig. 10). The incorporation of func- tional internalization peptides to lysine enriched ELR were necessary for higher transfection efﬁciency levels. Indeed, polyplexes containing the LAELIK120CPP polymer exhibited the highest luciferase expression with more than ﬁvefold than pDNA. Once the polyplexes are inside the endosome, LAEL seems to play a key role in leakage of the pDNA from the endosome. After incubation for 5 h, the luciferase pDNA delivered by LAELIK120CPP was able to reach the nucleus. In contrast, the main role of penetratin appears to be during cell entry and it is not as important as regards endosomal escape even though some plasmid still reaches the nucleus. Additionally, LAEL seems to promote slower but more efﬁ- cient outﬂow of the imidazole groups bound to the lysine- rich polymer. Hence, LAEL plays a more relevant role in transfection than CPP alone and incorporation of imidazole groups. Lower transfection efﬁciency for ELRs was found when comparing with PEI due to their lower charge density that may give to the ELRs more compatible features. Similar results of transfection had been previously reported for ELRs appended with oligo-lysine or p [Asp(DET)]53 in com- parison with BPEI, 61,62 where despite of its toxicity, PEI was also selected to compare luciferase expression levels due to its good transfection properties. 63,64 Taking together, the previous research of ELR as nonviral vectors showed higher toxicity than the ELRs developed in this work, with a viabil- ity up to 70% for the oligo-lysine modiﬁed ELR and from 80% for p [Asp(DET)]53. The ﬁrst was formed by an ELR- based diblock bearing an oligo-lysine for pDNA condensa- tion. The second was constituted by an ELR-based diblock containing diethylenetriamine (DET) modiﬁed poly- L -aspar-
Abstract: Diatoms are microalgae organisms that have a cover of silica, with a fascinating ordered porous structure that varies in size, giving them some different characteristics. Because of their different size, shape, and structure, it has incredible properties, letting them be capable of been functionalized with other particles. Therefore, due to the ordered pore structure, the high surface area, biocompatibility, availability, and low processing cost, they present a growing potential for drugdelivery when talking about silica materials, natural and synthetic, not to mention that is less expensive and a green alternative.
Cationic lipid based vesicles and polypeptides represent common non-viral delivery systems for in vitro and in vivo functional gene transfer for gene therapy purposes [1–5]. There exist a great variety of types of non-viral vectors [1, 6, 7]. They possess a number of advantages comparing to the viral vectors: they are not immunogenic like adenoviruses, not randomly integrated into genome like retro viruses, not infectious, not patogenic (oncogenic) and cheap. Neutral- izing DNA negative charge they facilitate adsorbic endocytosis of self-assembled complexes between plasmid DNA and polycation and/or cationic lipid particle – lipoplexes. Another possiblity for genosomes to be internalized is receptor-mediated endocytosis [8–10]. The most promissing approach to the latter mechanism of targeted gene transfer/delivery is to employ specific oligosaccharide-conjugated vector systems [11, 12]. Systems for targeted deliveryand receptor-mediated gene transfer could be also designed on the basis of polycations, but mainly using coupling with carbohydrates . Polycations conjugated with carbohy- drate residues were introduced into gene transfer field, and appeared to be one of the most effective group of transfection agents due to the moieties employed responsible for the receptor-mediated gene transfer [12, 13]. A number of chitosan preparations were recently reported as gene transfer anddelivery systems [14–16]. Galactose derivative of cholesterol was introduced to provide gene targeting to hepatocytes . In our study we emploied the incapsulation of reporter plasmid DNA into new delivery systems based on glycolipids, which are combining the advantages of both gene transfer mechanisms: non-specific (adsorbic endocy- tosis) and receptor-mediated ones, along with DNA incapsulation into hydrophobic oligocations.
SUMMARY . The purpose of the present study was to develop and optimize floating-bioadhesive bilayer gastroretentive drugdelivery system (GRDDS) exhibiting a unique combination of floatation and bioadhe- sion to prolong residence in the stomach using captopril (CP) and hydrochlorothiazide (HCTZ) as a model drug. Captopril being unstable in intestinal pH and HCTZ has specific absorption from duodenum and the first part of the jejunum and to a small extent in the stomach are suitable candidate for GRDDS. 3 2
The fibronectin type III domain (Fn3), which binds the integrin αvβ3, has also been fused to ELR diblocks . The self-assembly behaviour of the resulting constructs was studied through the bioproduction of ELRs with different hydrophilic and hydrophobic block lengths. The result was an increase in the uptake of ELR with Fn3 in comparison with the unimers in a transfected human leukaemia cell line overexpressing the integrin αvβ3 (K562/αvβ3). In a later study, a qualitative breakthrough in the design of ELR diblocks fused to functional peptides was accomplished. Thus, Chilkoti’s group constructed an ELR-based drug carrier termed nanopeptifier, that was able to improve cellular uptake by controlled exposure of CPPs, which toggled in response to the application of external mild hyperthermia (Figure 3) . The construct (Arg8-ELRBC- cBH3) was genetically engineered to possess a CPP (Arg8) at the N-terminus and the therapeutic payload peptide cBH3 (derived from the proapoptotic Bak protein) located at the C- terminus. The therapeutic cBH3 peptide was separated by an RVRR peptide linker cleavable by furin and cathepsin B proteases. The nanopeptifiers are soluble at physiological temperature (37 °C), whereas under of mild hyperthermia conditions (42 °C) they self-assemble into spherical micelles displaying a high density of CPP on their corona and with cBH3 located in the micelle core. Although some uptake of the nanopeptifier occurs at 37 °C, when the temperature is increased to 42 °C this cell uptake increases dramatically. Once the complex is internalized, the action of either furin (localized in the trans-Golgi) or cathepsin B (early and late endosomes and lysosomes) favours release of the cBH3 load. Increases of up to 130 arbitrary cell fluorescence units were found to induce the therapeutic effect of cBH3. Its therapeutic action was assessed by activation of caspase-3, which is involved in apoptotic processes. Thus, the nanopeptifier was able to create a cytotoxic switch that induces apoptosis only when it is in the micelle conformation.
The amino-terminal globular domain contains motifs in its link domain (amino acids 32-123) that bind HA and other glycosaminoglycans. The link domain contains four highly conserved cysteine residues that form interchain disulphide links that account for stability of the module. Outside the link motif there are two additional cysteine residues thought necessary for correct folding and GAG binding. The stem structure separates the amino-terminal domain from the transmembrane region and is consisted of 46 amino acids. It contains alleged proteolytic cleavage sites. The transmembrane region of CD44 consists of 23 hydrophobic amino acids and is thought to have a role in the grouping of CD44 in lipid rafts. The carboxy-terminal cytoplasmic domain binds proteins associated to signaling and cytoskeletal organization, such as ankyrin and ERM family proteins (ERM: ezrin, radixin and moesin). The cytoplasmic tail contains two serine residues the phosphorylation of which, and therefore binding of the proteins, is controlled by various kinases, including protein kinase C (PKC) and Rho kinase (Ponta et al., 2003).
SUMMARY . The self-microemulsifying drugdelivery system (SMEDDS) was employed to improve the bioavailability of sulpiride, a drug which is poorly soluble. The mean droplet size and emulsification time of the test formulation used for in vivo study were 9.27 ± 2.02 nm and 87 ± 5 s, respectively. When com- pared with Reference (Dogmatil®), the test formulation exhibited faster in-vitro drug release rate. The C max and AUC values of the test formulation were significantly higher than those of Reference, with an
Despite the success of the minimalist model 14b, self-immolative systems 1 and 2, developed by Dr. Sampedro, resulted synthetically tedious and low in yield. Additionally, the most important challenge to overcome was their low water solubility, which made them hard to study in simulated physiological conditions. For this reason, the subsequent design and development of disulfide-based SIL-drug conjugates should include cunning chemical modifications to enhance the solubility of the resulting structure which requires an additional synthetic handicap. Besides, it is worth to mention that the unexpected degradation of cyclothiosquaramide 24, the main product of such disassembly processes, hinders the spectroscopic study of the reaction kinetics. With such precedents, we decided to discard in this thesis, the design and development of disulfide-based SIL and focus on the potential of amino derivatives.
Metal-Organic Framework (MOF) are an emerging class of materials that have drown wide interest in many fields for applications like catalysis, sensing, nonlinear optics and potential biomedical applications as DDS . MOF consist in a class of hybrid materials obtained by self-assembly processes of metallic ions or clusters and organic ligands used to bridge them the metal components. The resulting structure is a three-dimensional network characterized by regular porosity. The virtually limitless combinations of metals and ligands allow to assemble structures with versatile architecture, morphology, size and physicochemical properties [154,155]. Their physicochemical properties remain unaffected by further modifications, maintaining uniformity of the obtained MOF. Additionally, MOF have large surface area and porosity, which provide high loading capacity, while the weak coordination bonds provide better biodegradation. A unique behaviour observed for MOF is their ability to adapt the pore opening to the dimensions of the drugs, in order to optimize drug-matrix interactions thus improving the loading capacity while controlling better the release time. Studies on MOFs based on iron, zinc and zirconium report on a significant increase in the release time for up to several weeks [156-158]. Furthermore, few reports show how the release can be triggered by several single or multiple stimuli, allowing to control the delivery of loaded molecules upon activation by variation in pH, magnetic field, ions, temperature, light and pressure. For example, Fe 2 O 3 -based MOF exhibited controlled
The ultimate goal for diabetes mellitus is to control the blood glucose level by insulin deliveryand minimized the long-term diabetic complications. Currently main therapy is to administered insulin through subcutaneous injection for diabetes patients. Two or three injections are required a day to maintain the normal blood glucose level. Because this method is burdensome and invasive to living organ- isms, the patient’s situation would not be good regarding the quality of life. Therefore, an electrical and mechanical controlled insulin pump that injects insulin automatical- ly into the bloodstream has been developed. An insulin pump constructed with polymer materials has been stud- ied. Many researchers working on deliver insulin by smart device.
In-vitro drug release of Isoniazid Loaded Microspheres A dialysis membrane was used to monitor Isoniazid release from the Microspheres. Dialysis membrane with molecu- lar weight cut off 12,000 KD was used to determine the re- lease of isoniazid from Microspheres. For determining the in-vitro drug release the 7.4 phosphate buffer solution was prepared. The dialysis membrane was soaked overnight in a buffer in order to open the pores of the membrane. The dialysis membrane was taken out from the buffer and was tied at one end. The Prepared Isoniazid Microspheres was introduced into the membrane and the other end was also tied with thread so that the sample should remain in the membrane. The membrane was introduced in the buffer medium. At frequent time intervals 1 ml aliquot was with- drawn and after sufficient dilution was analyzed spectro- photometrically at 262 nm. The sample Withdrawn stayed replaced via an equal quantity of fresh 7.4 phosphate Buffer saline. The drug release profile is presented graphically.
systems such as cell cultures, where the efficacy of free cisplatin is extremely high. Additionally, in the case of AuNPs-cisplatin, the vehicle protects the drug both from renal clearance and plasma deactivation because the strength of the link avoids unspecific release of cisplatin. However, the release of cisplatin from the NPs is sustained over time after the triggering. For example, the release at pH 4.4 was 10 % after 24 hours (as showed in chapter 4, figure 4.8). Thus, when same doses of free cisplatin and AuNPs- cisplatin are used in in vitro experiments, in which there are no loses of drug in any case, it is likely that in the AuNPs-cisplatin treatment, the low release of cisplatin from the AuNPs make the effective dose much lower than when treated with free drug. This is corroborated by the lower accumulation on the DNA after 24 hours. Fortunately, this is largely compensated in in vivo experiments, in which 98% of free cisplatin is rapidly lost either by renal clearance or by albumin deactivation, whilst the clearance of AuNPs- cisplatin is much more slow. Note also, that higher doses of AuNPs-cisplatin could not be used due to the impossibility to concentrate more the NPs and the restriction of the volume of treatment that can be added to cells, which is never higher than 10 % of total volume. This lack of efficacy in in vitro assays has been reported in other cases which showed later efficacy in in vivo experiments.
PEG 400 were dissolved in 50 ml of acetone and methanol mixture in ratio of 4:1. The coating operation was performed in a conventional laboratory model stainless steel spray coating pan with hot air blower. The speed of the pan was adjusted to 30 rpm. The inlet air temperature was 40- 45ºC and the coating was done manually by intermittent spraying and drying technique (4 ml/minute). Coated tablets were allowed to dry completely in a hot air oven at 60ºC for 12 hours. The weight of the tablet and thickness was determined before and after coating 12 .