Temperature/pH-Sensitive Double Cross-Linked Hydrogels as Platform for Controlled Delivery of Metoclopramide.

Novel double cross-linked (DC) hydrogels with pH-/temperature-sensitive properties were designed and developed. Therefore, linear pH-sensitive poly(methyl vinyl ether-alt-maleic acid) (P(VME/MA)) macromolecules were absorbed within a thermosensitive poly(N-isopropylacrylamide-co-hydroxyethylacrylamide)-hydrogel (PNH) and, subsequently, cross-linked together through a solvent-free thermal method. As a novelty, double cross-linked hydrogels were obtained from previously purified polymers in the absence of any solvent or cross-linking agent, which are generally harmful for the body. The new DC structures were characterized by FT-IR spectroscopy, SEM, swelling kinetic measurements, and mechanical tests. The resulting scaffolds exhibited interconnected pores and a flexible pattern, compared to the brittle structure of conventional PNH. The swelling kinetics of DC hydrogels were deeply affected by temperature (25 and 37 °C) and pH (7.4 and 1.2). Furthermore, the hydrogels absorbed a great amount of water in a basic environment and displayed improved mechanical properties. Metoclopramide (Met) was loaded within DC hydrogels as a model drug to investigate the ability of the support to control the drug release rate. The results obtained recommended them as convenient platforms for the oral administration of drugs, with the release of the largest part of the active principle occurring in the colon.

Bio-Responsive Carriers for Controlled Delivery of Doxorubicin to Cancer Cells.

The cellular internalization of drug carriers occurs via different endocytic pathways that ultimately involve the endosomes and the lysosomes, organelles where the pH value drops to 6.0 and 5.0, respectively. We aimed to design and characterize pH/temperature-responsive carriers for the effective delivery of the anti-tumoral drug doxorubicin. To this purpose, poly(N-isopropylacrylamide-co-vinylimidazole) was synthesized as an attractive pH/temperature-sensitive copolymer. Microspheres made of this copolymer, loaded with doxorubicin (MS-DXR), disintegrate in monodisperse nanospheres (NS-DXR) under conditions similar to that found in the bloodstream (pH = 7.4, temperature of 36 °C) releasing a small amount of payload. However, in environments that simulate the endosomal and lysosomal conditions, nanospheres solubilize, releasing the entire amount of drug. We followed the NS-DXR internalization using two cancer cell lines, hepatic carcinoma HepG2 cells and lung adenocarcinoma A549 cells. The data showed that NS-DXR are internalized to a greater extent by HepG2 cells than A549 cells, and this correlated with increased cytotoxicity induced by NS-DXR in HepG2 cells compared with A549 cells. Moreover, NS-DXR particles do not cause hemolysis and erythrocytes aggregation. Administered in vivo, NS-DXR localized in the liver and kidneys of mice, and the loading of DXR into NS resulted in the reduced renal clearance of DXR. In conclusion, the newly developed poly(N-isopropylacrylamide-co-vinyl imidazole) particles are biocompatible and may be introduced as carriers for doxorubicin to hepatic tumors.

PVA/Chitosan Thin Films Containing Silver Nanoparticles and Ibuprofen for the Treatment of Periodontal Disease.

Local delivery of drugs or antimicrobial agents is a suitable approach in the management of periodontitis when the infection is localized deep in the pockets and does not adequately respond to mechanical debridement and/or systemic antibiotic treatment. In this context, the objective of this study was to prepare new biocomposite films with antimicrobial, anti-inflammatory, and good mechanical properties to be applied in periodontal pockets. The composite film is eco-friendly synthesized from poly(vinyl alcohol) (PVA) cross-linked with oxidized chitosan (OxCS). Silver nanoparticles (AgNps) were inserted during film synthesis by adding freshly chitosan-capped AgNps colloidal solution to the polymer mixture; the addition of AgNps up to 1.44 wt.% improves the physico-chemical properties of the film. The characterization of the films was performed by FT-IR, atomic mass spectrometry, X-ray spectroscopy, and SEM. The films displayed a high swelling ratio (162%), suitable strength (1.46 MPa), and excellent mucoadhesive properties (0.6 N). Then, ibuprofen (IBF) was incorporated within the best film formulation, and the IBF-loaded PVA/OxCS-Ag films could deliver the drug in a sustained manner up to 72 h. The biocomposite films have good antimicrobial properties against representative pathogens for oral cavities. Moreover, the films are biocompatible, as demonstrated by in vitro tests on HDFa cell lines.

A novel platform for drug testing: Biomimetic three-dimensional hyaluronic acid-based scaffold seeded with human hepatocarcinoma cells.

Hepatic cancer is one of the most widespread maladies worldwide that requires urgent therapies and thus reliable means for testing anti-cancer drugs. The switch from two-dimensional (2D) to three-dimensional (3D) cell cultures produced an improvement in the in vitro outcomes for testing anti-cancer drugs. We aimed to develop a novel hyaluronic acid (HA)-based 3D cell model of human hepatocellular carcinoma (HepG2 cells) for drug testing and to assess comparatively in 3D vs. 2D, the cytotoxicity and the apoptotic response to the anti-tumor agent, cisplatin. The 3D model was developed by seeding HepG2 cells in a HA/poly(methylvinylether-alt-maleic acid) (HA3P50)-based scaffold. Compared to 2D, the cells grown in the HA3P50 scaffold proliferate into larger-cellular aggregates that exhibit liver-like functions by controlling the release of hepatocyte-specific biomarkers (albumin, urea, bile acids, transaminases) and the synthesis of cytochrome-P450 (CYP)7A1 enzyme. Also, growing the cells in the scaffold sensitize the hepatocytes to the anti-tumor effect of cisplatin, by a mechanism involving the activation of ERK/p38α-MAPK and dysregulation of NF-kB/STAT3/Bcl-2 pathways. In conclusion, the newly developed HA-based 3D model is suitable for chemotherapeutic drug testing on hepatocellular carcinoma. Moreover, the system can be adapted and employed as experimental platform functioning as a proper tissue/tumor surrogate.

Smart drug delivery system activated by specific biomolecules.

Essentially, the human body can release in different disease conditions specific biomolecules such as histamines when the body encounters a toxic substance, antibodies which are part of the body's natural immune response or nitric oxide as a cardiovascular signalling molecule. Design and development of "intelligent" delivery systems able to release the therapeutic agent only in the presence of bioactive compounds was presented here. Poly(N-isopropylacrylamide-co-N-(3-aminopropyl)methacrylamide)) (poly(NIPAAm-co-APM)) was synthesized as an exciting pH/temperature sensitive copolymer. Under physiological conditions (pH = 7.4), the APM in copolymer is in the ionized state (pKa = 8.7), highly hydrophilic and therefore the copolymer loses thermosensitive properties. Remarkably, after electrostatic interactions of APM with specific biomolecules, the copolymer restores the thermosensitive property. Thus, the microgels synthesized from this copolymer are in the "inactivated" state at normal physiological pH and temperature (pH = 7.4 and T = 36 °C). In the presence of specific biomolecules, microgels undergo "activation", shrink and expel mechanically a certain amount of drug. It must be mentioned that the pH-sensitive component plays the role of a biosensor, the biomolecule acts as a triggering agent, and the poly(NIPAAm) represents the delivery component (actuator). MTT tests showed that poly(NIPAAm-co-APM) microspheres are completely devoid of toxicity; moreover, the rabbit dermal fibroblasts vastly adhere to the surface of microspheres.

Pullulan derivative with cationic and hydrophobic moieties as an appropriate macromolecule in the synthesis of nanoparticles for drug delivery.

A new amphiphilic pullulan derivative (DBAP-PO) was obtained by grafting tertiary butyl amine and octanoyl groups on the pullulan backbone as cationic and hydrophobic moieties, respectively. The structural characteristics of the modified polymer were investigated by FT-IR and 1H and 13C NMR spectroscopy. The self-association ability in aqueous solution of DBAP-PO was studied by viscosity and fluorescence methods. The intrinsic viscosity of the polymer was determined by Wolf model. The critical aggregation concentration (CAC) value of 0.028 g/dL, determined by fluorescence measurements in the presence of pyrene, was confirmed by capillary viscosimetry and dynamic laser scattering (DLS). Dialysis method was used to demonstrate the capacity of the pullulan derivative to form spherical nanoparticles (d ~ 200 nm) loaded with model drug, sodium diclofenac (DF) (74% entrapment efficiency). The DF release was sustained and pH-dependent. In vitro cytotoxicity as well as morphological studies conducted on the human skin fibroblasts showed that DBAP-PO/DF nanoparticles do not exhibit cytotoxic effects at the pharmacologically relevant concentration of DF, maintaining the typical morphology of the cells.

A new sponge-type hydrogel based on hyaluronic acid and poly(methylvinylether-alt-maleic acid) as a 3D platform for tumor cell growth.

A new sponge-type hydrogel was obtained by cross-linking hyaluronic acid (HA) and poly(methylvinylether-alt-maleic acid) P(MVE-alt-MA) through a solvent-free thermal method. The sponge-type hydrogel was characterized and checked as a support for cell growth. The influence of concentration and weight ratio of polymers on the morphology and hydrogel stability was investigated. The total polymers concentration of 3% (w/w) and the weight ratio of 1:1 were optimal for the synthesis of a stable hydrogel (HA3P50) and to promote cell proliferation. The swelling measurements revealed a high-water absorption capacity of the hydrogel in basic medium. Diphenhydramine (DPH), lidocaine (Lid) and propranolol (Prop) were loaded within the hydrogel as a model drugs to investigate the ability of drug transport and release. In vitro studies revealed that HA3P50 hydrogel promoted the adhesion and proliferation of human hepatocellular carcinoma cell line HepG2, providing a good support for 3D cell culture to obtain surrogate tumor scaffold suitable for preclinical anti-cancer drug screening.

Simple and dual cross-linked chitosan millicapsules as a particulate support for cell culture.

The chitosan hydrochloride (Cs·HCl) was obtained as a polymer soluble in physiological solutions to be used as potential support for safely cell culture or cell encapsulation. Viability tests showed that concentrations between 0.16 and 5 mg/mL of Cs·HCl were not toxic for the HEK293 cells. In parallel, aldehyde-functionalized pullulan (Pul-CHO) was synthesized as the macromolecular cross-linker. Cs·HCl was dissolved in 0.9% NaCl and injected (INJECTOMAT SEP 21S PLUS) through a needle to obtain small droplets in a sodium tripolyphosphate solution in the absence and presence of 0.1% (w/v) Pul-CHO. Simple and dual cross-linked millicapsules were obtained with pore size ranging from 50 µm to 5 µm, respectively. FITC-Dextran with molecular weights of 4000 and 70,000 g/mol was encapsulated during microcapsule synthesis as macromolecular models to check the permeability of Cs millicapsules. The results show that FITC-Dextran 4000 and 70,000 diffuses quickly from simple cross-linked millicapsules while dual cross-linked millicapsules release slowly both FITC-dextrans. Microscopy experiments show that HEK 293 cells adhere to the surface of millicapsules. Taken together the data reveal that Cs millicapsules allow the cell growth on their surface, and thus, they offer new perspectives for cell encapsulation strategy.

Smart composite materials based on chitosan microspheres embedded in thermosensitive hydrogel for controlled delivery of drugs.

Smart composite hydrogels (SCHs) consisting of chitosan (CS) microspheres physically embedded within a thermoresponsive hydrogel are synthesized and tested for their capacity of loading and long-term release of a small molecule drug. CS microspheres were used since they display pH-sensitive properties and have the capacity to bind electrostatically the opposite charged salicylic acid (SA), taken as model drug. These microspheres are ulterior physically entrapped within a thermoresponsive hydrogel based on poly(N-isopropylacrylamide-co-hydroxyethylacrylamide) copolymer, cross-linked with N,N'-methylenebisacrylamide. The morphology, swelling behavior, temperature and pH sensitivity, degradability and drug release behavior of the new smart drug delivery system were investigated. Swelling ratios as well as the sharpness of the phase transition, largely depended on the cross-linking degree. The thermoresponsive network slightly protected the CS microspheres from the in vitro degradation. In vitro studies showed that the SA followed a prolonged release profile from SCHs in accordance with pH and temperature.

Smart nanoparticles based on pullulan-g-poly(N-isopropylacrylamide) for controlled delivery of indomethacin.

Double hydrophilic thermo-responsive pullulan-g-poly(N-isopropylacrylamide) (P-g-pNIPAM) copolymers with two different molecular weight of thermosensitive grafts were synthesized and used for preparation of indomethacin-loaded nanoparticles by dialysis and nanoprecipitation method. The polymers form aggregates in aqueous solution at a concentration of 10g/L, above their critical aggregation concentration (3.36g/L) and below the lower critical solution temperature (LCST). After indomethacin loading, nanoparticles with compact and uniform structure were formed below the LCST. The effects of copolymer composition, concentration, and the feed polymer/drug ratio on the particle size, drug loading content (DLC) and entrapment efficiency (EE) were investigated. DLC increased with drug feeding, reaching a maximum value of 40% at the ratios of 1/1. Smaller particles (145nm) with narrower size distribution were obtained from polymer with a higher molecular weight of pNIPAM grafts. FT-IR and 1H NMR spectra proved that the main driven force for the aggregation was the hydrogen bonding between indomethacin and the pNIPAM side chains of copolymer. The indomethacin release rate from nanoparticles was influenced by temperature, because of the dissociation of the hydrogen bonds at high temperatures, the degree of drug loading, and the pH of the release media.

Do cyclodextrins bound to dextran microspheres act as sustained delivery systems of drugs?

The use of cyclodextrins (CDs) for controlled delivery of drugs is largely presented in the literature. However, the question of whether CDs themselves linked to a polymeric network are able to sustain the release of drugs still persists. Here, CD immobilization within dextran microspheres is reported, and CD-dextran complexes were packed in a glass column and then, the retention time of different drugs and drug model compounds was determined by liquid chromatography. The release profiles of drugs and of drug model compounds (indole, 3-nitrophenol, p-hydroxybenzoic acid, diclofenac), characterized by different values of the retention time (high, moderate or low), were investigated. The release rates were quite high even for drugs that exhibit very high retention time (high association equilibrium constant). Moreover, the volume of the release fluid strongly influences the rate of drug release. As a whole, "the sink conditions" must be continuously maintained, since at each drug concentration in the release medium, equilibrium occurs between the free and the CD-bound drug.

Poly(N-isopropylacrylamide-co-methacrylic acid) pH/thermo-responsive porous hydrogels as self-regulated drug delivery system.

A dual drug delivery system based on a "biosensor" (pH-sensitive unit) and a delivery component (thermosensitive hydrogel) was developed. The pH/thermosensitive hydrogel is able to restore the thermosensitive characteristics after electrostatic interaction of the pH-sensitive units with selected biologically active compounds that act as triggering agents. The poly(N-isopropylacrylamide-co-methacrylic acid) (poly(NIPAAm-co-MA)) was synthesized as an interesting pH/thermo-responsive copolymer by free radical polymerization method. Due to the presence of carboxylic groups in MA units, the copolymer loses its thermosensitivity at physiological pH and temperature. However, when the negatively-charged carboxylic groups of the pH-sensitive units interact electrostatically with the positively-charged drugs with hydrophobic character propranolol, lidocaine or metoclopramide, taken as model biologically active compounds, the copolymer restores the thermosensitive properties around the physiological pH and temperature. The poly(NIPAAm-co-MA) linear copolymer was converted into pH/thermo-responsive porous microgels using oligomers of NIPAAm above their LCST, as porogens. Accordingly, the swelling/collapsing processes of the microgels occur only after the interaction with the positively-charged hydrophobic drugs. The hydrophobic drug acts as a triggering agent and the pH/temperature sensitive hydrogel turns as a biosensor (pH-sensitive units) and a delivery component (thermosensitive hydrogel).

Poly(N-isopropylacrylamide-co-hydroxyethylacrylamide) thermosensitive microspheres: the size of microgels dictates the pulsatile release mechanism.

Poly(N-isopropylacrylamide-co-N-hydroxyethylacrylamide) (poly(NIPAAm-co-HEAAm)) was prepared as a new thermosensitive copolymer possessing a sharp phase transition around the human body temperature. The effect of the copolymer concentration on the lower critical solution temperature (LCST) was determined under physiological conditions by cloud point (CP) and differential scanning calorimetric (DSC) methods. Then, thermosensitive microspheres were prepared from preformed copolymers by chemical cross-linking of hydroxyl groups with glutaraldehyde at a temperature situated slightly below LCST of the copolymer solution. The volume phase transition temperature (VPTT) of corresponding cross-linked microspheres was determined from swelling degree-temperature curve. The microspheres were loaded with model drug indomethacin by the solvent evaporation method. The DSC analysis proved that the drug is molecularly dispersed in the polymer network. Finally, the influence of the microsphere size on drug release was investigated. It was established that microspheres with the diameter ranging between 5 and 60 µm release the drug with almost the same rate below (in the swollen state) and above the VPTT (in the collapsed state). On the contrary, microspheres with the diameter ranging between 125 and 220 µm release a significantly higher amount of indomethacin below than above the VPTT. This different behavior is enough to assure a pulsatile release mechanism when the temperature changes cyclically below and above the VPTT. However, both small and large microspheres release a large amount of the drug during the collapsing process.