3D printing of gellan-dextran methacrylate IPNs in glycerol and their bioadhesion by RGD derivatives.

The ever-growing need for new tissue and organ replacement approaches paved the way for tissue engineering. Successful tissue regeneration requires an appropriate scaffold, which allows cell adhesion and provides mechanical support during tissue repair. In this light, an interpenetrating polymer network (IPN) system based on biocompatible polysaccharides, dextran (Dex) and gellan (Ge), was designed and proposed as a surface that facilitates cell adhesion in tissue engineering applications. The new matrix was developed in glycerol, an unconventional solvent, before the chemical functionalization of the polymer backbone, which provides the system with enhanced properties, such as increased stiffness and bioadhesiveness. Dex was modified introducing methacrylic groups, which are known to be sensitive to UV light. At the same time, Ge was functionalized with RGD moieties, known as promoters for cell adhesion. The printability of the systems was evaluated by exploiting the ability of glycerol to act as a co-initiator in the process, speeding up the kinetics of crosslinking. Following semi-IPNs formation, the solvent was removed by extensive solvent exchange with HEPES and CaCl2, leading to conversion into IPNs due to the ionic gelation of Ge chains. Mechanical properties were investigated and IPNs ability to promote osteoblasts adhesion was evaluated on thin-layer, 3D-printed disk films. Our results show a significant increase in adhesion on hydrogels decorated with RGD moieties, where osteoblasts adopted the spindle-shaped morphology typical of adherent mesenchymal cells. Our findings support the use of RGD-decorated Ge/Dex IPNs as new matrices able to support and facilitate cell adhesion in the perspective of bone tissue regeneration.

Polysaccharide-Based Nanogels to Overcome Mucus, Skin, Cornea, and Blood-Brain Barriers: A Review.

Nanocarriers have been extensively developed in the biomedical field to enhance the treatment of various diseases. However, to effectively deliver therapeutic agents to desired target tissues and enhance their pharmacological activity, these nanocarriers must overcome biological barriers, such as mucus gel, skin, cornea, and blood-brain barriers. Polysaccharides possess qualities such as excellent biocompatibility, biodegradability, unique biological properties, and good accessibility, making them ideal materials for constructing drug delivery carriers. Nanogels, as a novel drug delivery platform, consist of three-dimensional polymer networks at the nanoscale, offering a promising strategy for encapsulating different pharmaceutical agents, prolonging retention time, and enhancing penetration. These attractive properties offer great potential for the utilization of polysaccharide-based nanogels as drug delivery systems to overcome biological barriers. Hence, this review discusses the properties of various barriers and the associated constraints, followed by summarizing the most recent development of polysaccharide-based nanogels in drug delivery to overcome biological barriers. It is expected to provide inspiration and motivation for better design and development of polysaccharide-based drug delivery systems to enhance bioavailability and efficacy while minimizing side effects.

Polyacrylate-Cholesterol Amphiphilic Derivative: Formulation Development and Scale-up for Health Care Applications.

The novel amphiphilic polyacrylate grafted with cholesterol moieties, PAAbCH, previously synthesized, was deeply characterized and investigated in the lab and on a pre-industrial scale. Solid-state NMR analysis confirmed the polymer structure, and several water-based pharmaceutical and cosmetic products were developed. In particular, stable oil/water emulsions with vegetable oils, squalene, and ceramides were prepared, as well as hydrophilic medicated films loaded with diclofenac, providing a prolonged drug release. PAAbCH also formed polyelectrolyte hydrogel complexes with chitosan, both at the macro- and nano-scale. The results demonstrate that this polymer has promising potential as an innovative excipient, acting as a solubility enhancer, viscosity enhancer, and emulsifying agent with an easy scale-up transfer process.

Therapeutic effects of dexamethasone-loaded hyaluronan nanogels in the experimental cholestasis.

A major function of the intrahepatic biliary epithelium is bicarbonate excretion in bile. Recent reports indicate that budesonide, a corticosteroid with high receptor affinity and hepatic first pass clearance, increases the efficacy of ursodeoxycholic acid, a choleretic agent, in primary biliary cholangitis patients. We have previously reported that bile ducts isolated from rats treated with dexamethasone or budesonide showed an enhanced activity of the Na+/H+ exchanger isoform 1 (NHE1) and Cl-/HCO3- exchanger protein 2 (AE2) . Increasing the delivery of steroids to the liver may result in three beneficial effects: increase in the choleresis, treatment of the autoimmune or inflammatory liver injury and reduction of steroids' systemic harmful effects. In this study, the steroid dexamethasone was loaded into nanohydrogels (or nanogels, NHs), in order to investigate corticosteroid-induced increased activities of transport processes driving bicarbonate excretion in the biliary epithelium (NHE-1 isoform) and to evaluate the effects of dexamethasone-loaded NHs (NHs/dex) on liver injury induced by experimental cholestatis. Our results showed that NHs and NHs/dex do not reduce cell viability in vitro in human cholangiocyte cell lines. Primary and immortalized human cholangiocytes treated with NHs/dex show an increase in the functional marker expression of NHE1 cholangiocytes compared to control groups. A mouse model of cholangiopathy treated with NHs/dex shows a reduction in markers of hepatocellular injury compared to control groups (NHs, dex, or sham group). In conclusion, we believe that the NHs/dex formulation is a suitable candidate to be investigated in preclinical models of cholangiopathies.

Hyaluronan-Cholesterol Nanogels for the Enhancement of the Ocular Delivery of Therapeutics.

The anatomy and physiology of the eye strongly limit the bioavailability of locally administered drugs. The entrapment of therapeutics into nanocarriers represents an effective strategy for the topical treatment of several ocular disorders, as they may protect the embedded molecules, enabling drug residence on the ocular surface and/or its penetration into different ocular compartments. The present work shows the activity of hyaluronan-cholesterol nanogels (NHs) as ocular permeation enhancers. Thanks to their bioadhesive properties, NHs firmly interact with the superficial corneal epithelium, without penetrating the stroma, thus modifying the transcorneal penetration of loaded therapeutics. Ex vivo transcorneal permeation experiments show that the permeation of hydrophilic drugs (i.e., tobramycin and diclofenac sodium salt), loaded in NHs, is significantly enhanced when compared to the free drug solutions. On the other side, the permeation of hydrophobic drugs (i.e., dexamethasone and piroxicam) is strongly dependent on the water solubility of the entrapped molecules. The obtained results suggest that NHs formulations can improve the ocular bioavailability of the instilled drugs by increasing their preocular retention time (hydrophobic drugs) or facilitating their permeation (hydrophilic drugs), thus opening the route for the application of HA-based NHs in the treatment of both anterior and posterior eye segment diseases.

Hyaluronic Acid Derivative Effect on Niosomal Coating and Interaction with Cellular Mimetic Membranes.

Hyaluronic acid (HA) is one of the most used biopolymers in the development of drug delivery systems, due to its biocompatibility, biodegradability, non-immunogenicity and intrinsic-targeting properties. HA specifically binds to CD44; this property combined to the EPR effect could provide an option for reinforced active tumor targeting by nanocarriers, improving drug uptake by the cancer cells via the HA-CD44 receptor-mediated endocytosis pathway. Moreover, HA can be easily chemically modified to tailor its physico-chemical properties in view of specific applications. The derivatization with cholesterol confers to HA an amphiphilic character, and then the ability of anchoring to niosomes. HA-Chol was then used to coat Span® or Tween® niosomes providing them with an intrinsic targeting shell. The nanocarrier physico-chemical properties were analyzed in terms of hydrodynamic diameter, ζ-potential, and bilayer structural features to evaluate the difference between naked and HA-coated niosomes. Niosomes stability was evaluated over time and in bovine serum. Moreover, interaction properties of HA-coated nanovesicles with model membranes, namely liposomes, were studied, to obtain insights on their interaction behavior with biological membranes in future experiments. The obtained coated systems showed good chemical physical features and represent a good opportunity to carry out active targeting strategies.

Nano-hydrogel embedded with quercetin and oleic acid as a new formulation in the treatment of diabetic foot ulcer: A pilot study.

Wound healing, especially diabetic ones, is a relevant clinical problem, so it is not surprising that surgical procedures are often needed. To overcome invasive procedures, several strategies with drugs or natural compound are used. Recently, in an experimental study, we described an increase in keratinocyte proliferation after their exposition to quercetin plus oleic acid. In the present clinical study, we evaluated both the clinical efficacy and the safety of nano-hydrogel embedded with quercetin and oleic acid in the treatment of lower limb skin wound in patients with diabetes mellitus (DM). Fifty-six DM patients (28 men and 28 women, mean age 61.7 ± 9.2 years) unsuccessfully treated with mechanical compression were enrolled and randomised to receive an add on treatment with hyaluronic acid (0.2%) or nano-hydrogel embedded with quercetin and oleic acid. The treatment with nano-hydrogel embedded with quercetin and oleic acid significantly (P < .01) reduced the wound healing time, in comparison to hyaluronic acid (0.2%) without developing of adverse drug reactions, suggesting that this formulation could be used in the management of wound healing even if other clinical trials must be performed in order to validate this observation.

Intracellular Delivery of Natural Antioxidants via Hyaluronan Nanohydrogels.

Natural antioxidants, such as astaxanthin (AX), resveratrol (RV) and curcumin (CU), are bioactive molecules that show a number of therapeutic effects. However, their applications are remarkably limited by their poor water solubility, physico-chemical instability and low bioavailability. In the present work, it is shown that self-assembled hyaluronan (HA)-based nanohydrogels (NHs) are taken up by endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs), preferentially accumulating in the perinuclear area of oxidatively stressed HUVECs, as evidenced by flow cytometry and confocal microscopy analyses. Furthermore, NHs are able to physically entrap and to significantly enhance the apparent water solubility of AX, RV and CU in aqueous media. AX/NHs, RV/NHs and CU/NHs systems showed good hydrodynamic diameters (287, 214 and 267 nm, respectively), suitable ζ-potential values (-45, -43 and -37 mV, respectively) and the capability to neutralise reactive oxygen species (ROS) in tube. AX/NHs system was also able to neutralise ROS in vitro and did not show any toxicity against HUVECs. This research suggests that HA-based NHs can represent a kind of nano-carrier suitable for the intracellular delivery of antioxidant agents, for the treatment of oxidative stress in endothelial cells.

Long-Circulating Hyaluronan-Based Nanohydrogels as Carriers of Hydrophobic Drugs.

Nanohydrogels based on natural polymers, such as polysaccharides, are gaining interest as vehicles for therapeutic agents, as they can modify the pharmacokinetics and pharmacodynamics of the carried drugs. In this work, hyaluronan-riboflavin nanohydrogels were tested in vivo in healthy rats highlighting their lack of toxicity, even at high doses, and their different biodistribution with respect to that of native hyaluronan. They were also exploited as carriers of a hydrophobic model drug, the anti-inflammatory piroxicam, that was physically embedded within the nanohydrogels by an autoclave treatment. The nanoformulation was tested by intravenous administration showing an improvement of the pharmacokinetic parameters of the molecule. The obtained results indicate that hyaluronan-based self-assembled nanohydrogels are suitable systems for low-soluble drug administration, by increasing the dose as well as the circulation time of poorly available therapeutic agents.

Pursuing Intracellular Pathogens with Hyaluronan. From a 'Pro-Infection' Polymer to a Biomaterial for 'Trojan Horse' Systems.

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is 'Cluster of Differentiation 44' (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as 'reservoirs' for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a 'Trojan Horse'. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

Hyaluronan-Based Nanohydrogels for Targeting Intracellular S. Aureus in Human Keratinocytes.

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan-cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA-based NHs for treatment of such skin infections.

Preparation of gellan-cholesterol nanohydrogels embedding baicalin and evaluation of their wound healing activity.

In the present work, the preparation, characterization and therapeutic potential of baicalin-loaded nanohydrogels are reported. The nanohydrogels were prepared by sonicating (S nanohydrogel) or autoclaving (A nanohydrogel) a dispersion of cholesterol-derivatized gellan in phosphate buffer. The nanohydrogel obtained by autoclave treatment showed the most promising results: smaller particles (∼362 nm vs. ∼530 nm), higher homogeneity (polydispersity index = ∼0.24 vs. ∼0.47), and lower viscosity than those obtained by sonication. In vitro studies demonstrated the ability of the nanohydrogels to favour the deposition of baicalin in the epidermis. A high biocompatibility was found for baicalin-loaded nanohydrogels, along with a great ability to counteract the toxic effect induced by hydrogen peroxide in cells, as the nanohydrogels re-established the normal conditions (∼100% viability). Further, the potential of baicalin-loaded nanohydrogels in skin wound healing was demonstrated in vivo in mice by complete skin restoration and inhibition of specific inflammatory markers (i.e., myeloperoxidase, tumor necrosis factor-α, and oedema).

Gellan Nanohydrogels: Novel Nanodelivery Systems for Cutaneous Administration of Piroxicam.

The feasibility to use gellan nanohydrogels (Ge-NHs) as delivery system for the cutaneous administration of piroxicam (PRX) was investigated using gellan conjugated with cholesterol or riboflavin. The in vitro skin penetration studies through human epidermis were performed using a saturated aqueous drug solution, a 50% w/v Transcutol aqueous solution, and a commercially available PRX plaster as controls. Confocal microscopy, ATR-FTIR spectroscopy, circular dichroism, and a dynamometer assisted extrusion assay were performed to clarify the permeation mechanism of Ge-NHs. The skin permeation studies evidenced that Ge-NHs enhance the PRX retention in the epidermis and, at the same time, slow down the permeation process with respect to the controls. NHs can penetrate the stratum corneum, and then gradually disassemble thus diffusing in the viable epidermis reaching the spinosum layer. In conclusion, NHs represent a novel strategy to target poorly permeable compounds in the epidermis, thus improving the management of cutaneous pathologies.

"Click" hyaluronan based nanohydrogels as multifunctionalizable carriers for hydrophobic drugs.

Highly hydrophilic and biocompatible nanocarriers based on polysaccharide hydrogels (nanohydrogels, NHs) were shown to be promising systems for drug delivery applications. Following the idea of these emerging drug carriers, the aim of the present work was to develop self-assembled hydrogel nanoparticles based on amphiphilic derivatives of hyaluronic acid (HA) and riboflavin (Rfv), synthesized by "click" Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction. The obtained amphiphilic product (HA-c-Rfv) was able to form nanohydrogels in aqueous environments, in particular by applying an innovative autoclave-based method. HA of different molecular weights (Mw) and degrees of substitution (DS) were prepared and the effect of these parameters on the NHs formation was assessed. The derivative HA220-c-Rfv 40/40 was chosen as the most interesting system, capable to form NHs in the range of 150-200nm and with a negative ζ-potential. NHs were very stable in water solutions and, by adding dextrose as cryoprotectant, it was also possible to freeze-dry the NHs formulation. The developed system is proposed for the delivery of hydrophobic drugs; for this purpose, dexamethasone, piroxicam and paclitaxel were used as model drugs; these molecules were loaded into NHs with high efficiency by film-hydration technique. Furthermore, a HA-c-Rfv derivative bearing an excess of propargylic portions was capable to react with other N3-derivatized molecules, opening the route to a wide spectrum of functionalization opportunities: in this direction, PEG-N3 has been tested as a model molecule for the preparation of PEGylated NHs.

The Influence of pH on the Scleroglucan and Scleroglucan/Borax Systems.

The effects that an increase of environmental pH has on the triple helix of scleroglucan (Sclg) and on the Sclg/borax hydrogel are reported. Rheological experiments show that the hydrogel is less sensitive to pH increase than Sclg alone, while at pH = 14 a dramatic viscosity decrease takes place for both systems. This effect is evidenced also by the reduced water uptake and anisotropic elongation detected, at pH = 14, by the swelling behaviour of tablets prepared with the Sclg/borax system. On the opposite, a different behaviour was observed with guar gum and locust bean gum tablets, tested as reference polysaccharides. The effect of pH on the structure of Sclg and Sclg/borax was investigated also by means of spectroscopic approaches based on the interaction between Congo red (CR) and the Sclg triple helix. Obtained results indicated that the CR absorbance maximum is shifted as a function of pH and by the presence of borax. Principal component analysis allowed very precise identification of the pH value at which the Sclg helix collapses. Molecular dynamics simulations of the Sclg/borax-CR complex indicated that, at physiological pH, only a few ordered configurations are populated, according to the induced circular dichroism (CD) spectrum evidence.

Design of Hybrid Gels Based on Gellan-Cholesterol Derivative and P90G Liposomes for Drug Depot Applications.

Gels are extensively studied in the drug delivery field because of their potential benefits in therapeutics. Depot gel systems fall in this area, and the interest in their development has been focused on long-lasting, biocompatible, and resorbable delivery devices. The present work describes a new class of hybrid gels that stem from the interaction between liposomes based on P90G phospholipid and the cholesterol derivative of the polysaccharide gellan. The mechanical properties of these gels and the delivery profiles of the anti-inflammatory model drug diclofenac embedded in such systems confirmed the suitability of these hybrid gels as a good candidate for drug depot applications.

Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase.

Although in recent years several methods have been studied and developed to obtain different types of nanosized drug delivery systems, the set up of suitable procedures and materials remains highly expensive, their preparation is time consuming and often not feasible for a scale-up process. Furthermore, the sterilisation and storage of nanocarrier formulations represents a complicated but mandatory step for their effective use. In our previous work we assessed the use of an autoclaving process to achieve, in one simple step, sterile self-assembled hyaluronan-cholesterol (HA-CH) and hyaluronan-riboflavin (HA-Rfv) nanohydrogels (NHs). In the present work, the effect of the high temperature on HA-CH has been studied in detail. HA-CH suspensions were characterised in terms of size and polydispersity by Dynamic Light Scattering at different temperatures and conditions; the HA-CH chemical structure and its molecular weight were assessed via FT-IR and GPC analysis after the sterilising cycle in an autoclave at 121°C for 20min. The obtained NHs were then observed with TEM and AFM microscopy, in both dry and liquid conditions. The Young's modulus of the NHs was determined, evidencing the soft nature of these nanosystems; the critical aggregation concentration (c.a.c) of the nanosuspension was also assessed. Thereafter, alginate lyase (AL) was conjugated to NHs, with the aim of developing a useful system for therapies against bacterial infections producing alginate biofilms. The conjugation efficiency and the enzymatic activity of AL were determined after immobilisation. The AL-NHs system showed the ability to depolymerise alginate, offering an opportunity to be a useful nanosystem for the treatment of biofilm-associated infections.

One-step formation and sterilization of gellan and hyaluronan nanohydrogels using autoclave.

The sterilization of nanoparticles for biomedical applications is one of the challenges that must be faced in the development of nanoparticulate systems. Usually, autoclave sterilization cannot be applied because of stability concerns when polymeric nanoparticles are involved. This paper describes an innovative method which allows to obtain, using a single step autoclave procedure, the preparation and, at the same time, the sterilization of self-assembling nanohydrogels (NHs) obtained with cholesterol-derivatized gellan and hyaluronic acid. Moreover, by using this approach, NHs, while formed in the autoclave, can be easily loaded with drugs. The obtained NHs dispersion can be lyophilized in the presence of a cryoprotectant, leading to the original NHs after re-dispersion in water.

Polyaspartamide-doxorubicin conjugate as potential prodrug for anticancer therapy.

PURPOSE: To synthesize a new polymeric prodrug based on α,ß-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-d,l-aspartamide copolymer bearing amine groups in the side chain (PHEA-EDA), covalently linked to the anticancer drug doxorubicin and to test its potential application in anticancer therapy. METHODS: The drug was previously derivatized with a biocompatible and hydrophilic linker, leading to a doxorubicin derivative highly reactive with amino groups of PHEA-EDA. The PHEA-EDA-DOXO prodrug was characterized in terms of chemical stability. The pharmacokinetics, biodistribution and cytotoxicity of the product was investigated in vitro and in vivo on human breast cancer MCF-7 and T47D cell lines and NOD-SCID mice bearing a MCF-7 human breast carcinoma xenograft. Data collected were compared to those obtained using free doxorubicin. RESULTS: The final polymeric product is water soluble and easily hydrolysable in vivo, due to the presence of ester and amide bonds along the spacer between the drug and the polymeric backbone. In vitro tests showed a retarded cytotoxic effect on tumor cells, whereas a significant improvement of the in vivo antitumor activity of PHEA-EDA-DOXO and a survival advantage of the treated NOD-SCID mice was evidenced, compared to that of free doxorubicin. CONCLUSIONS: The features of the PHEA-EDA-DOXO provide a potential protection of the drug from the plasmatic enzymatic degradation and clearance, an improvement of the blood pharmacokinetic parameters and a suitable body biodistribution. The data collected support the promising rationale of the proposed macromolecular prodrug PHEA-EDA-DOXO for further potential development and application in the treatment of solid cancer diseases.

Highly versatile nanohydrogel platform based on riboflavin-polysaccharide derivatives useful in the development of intrinsically fluorescent and cytocompatible drug carriers.

In this work we describe a new nanohydrogel platform, based on polysaccharides modified with the hydrophobic and fluorescent molecule riboflavin tetrabutyrate, which leads to innovative structures useful for drug delivery applications. Hyaluronic acid and pullulan were chosen as representative of anionic and neutral polysaccharides, respectively, and the bromohexyl derivative of riboflavin tetrabutyrate was chemically linked to these polymer chains. Because of such derivatization, polymer chains were able to self-assemble in aqueous environment thus forming nanohydrogels, with mean diameters of about 312 and 210 nm, for hyaluronan and pullulan, respectively. These new nanohydrogels showed low polydispersity index, and negative ζ-potential. Moreover, the nanohydrogels, which can be easily loaded with model drugs, showed long-term stability in water and physiological conditions and excellent cytocompatibility. All these properties allow to consider these intrinsically fluorescent nanohydrogels suitable for the formulation of innovative drug dosage forms.