Mussel-Inspired Catechol Functionalisation as a Strategy to Enhance Biomaterial Adhesion: A Systematic Review.

Biomaterials have long been explored in regenerative medicine strategies for the repair or replacement of damaged organs and tissues, due to their biocompatibility, versatile physicochemical properties and tuneable mechanical cues capable of matching those of native tissues. However, poor adhesion under wet conditions (such as those found in tissues) has thus far limited their wider application. Indeed, despite its favourable physicochemical properties, facile gelation and biocompatibility, gellan gum (GG)-based hydrogels lack the tissue adhesiveness required for effective clinical use. Aiming at assessing whether substitution of GG by dopamine (DA) could be a suitable approach to overcome this problem, database searches were conducted on PubMed® and Embase® up to 2 March 2021, for studies using biomaterials covalently modified with a catechol-containing substituent conferring improved adhesion properties. In this regard, a total of 47 reports (out of 700 manuscripts, ~6.7%) were found to comply with the search/selection criteria, the majority of which (34/47, ~72%) were describing the modification of natural polymers, such as chitosan (11/47, ~23%) and hyaluronic acid (6/47, ~13%); conjugation of dopamine (as catechol "donor") via carbodiimide coupling chemistry was also predominant. Importantly, modification with DA did not impact the biocompatibility and mechanical properties of the biomaterials and resulting hydrogels. Overall, there is ample evidence in the literature that the bioinspired substitution of polymers of natural and synthetic origin by DA or other catechol moieties greatly improves adhesion to biological tissues (and other inorganic surfaces).

Lipid-based Nanocarriers for siRNA Delivery: Challenges, Strategies and the Lessons Learned from the DODAX: MO Liposomal System.

The possibility of using the RNA interference (RNAi) mechanisms in gene therapy was one of the scientific breakthroughs of the last century. Despite the extraordinary therapeutic potential of this approach, the need for an efficient gene carrier is hampering the translation of the RNAi technology to the clinical setting. Although a diversity of nanocarriers has been described, liposomes continue to be one of the most attractive siRNA vehicles due to their relatively low toxicity, facilitated siRNA complexation, high transfection efficiency and enhanced pharmacokinetic properties. This review focuses on RNAi as a therapeutic approach, the challenges to its application, namely the nucleic acids' delivery process, and current strategies to improve therapeutic efficacy. Additionally, lipid-based nanocarriers are described, and lessons learned from the relation between biophysical properties and biological performance of the dioctadecyldimethylammonium:monoolein (DODAX: MO) system are explored. Liposomes show great potential as siRNA delivery systems, being safe nanocarriers to protect nucleic acids in circulation, extend their half-life time, target specific cells and reduce off-target effects. Nevertheless, several issues related to delivery must be overcome before RNAi therapies reach their full potential, namely target-cell specificity and endosomal escape. Understanding the relationship between biophysical properties and biological performance is an essential step in the gene therapy field.

Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells.

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical conditions, siRNA-nanocarriers' stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers' efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers' behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipoplexes were found to have better colloidal stability as they could maintain a relatively stable size. In addition, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells. Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good colloidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. STATEMENT OF SIGNIFICANCE: This work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vectors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.

Tunable pDNA/DODAB:MO lipoplexes: the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency.

Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-ß-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODAB's tendency to form bilayer structures and MO's propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MO's structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increase in the incubation temperature has a similar effect on aggregate morphology as the observed with an increase in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphology and subsequent transfection efficiency defining new strategies in lipoplexes preparation that could be used to modulate the properties of other lipid formulations for nonviral gene delivery applications.