Targeted Nanoparticles Based on Alendronate Polyethylene Glycol Conjugated Chitosan for the Delivery of siRNA and Curcumin for Bone Metastasized Breast Cancer Applications.

Bone metastasized breast cancer reduces the quality of life and median survival. Targeted delivery of small interfering RNA (siRNA) and chemotherapeutic drugs using nanoparticles (NPs) is a promising strategy to overcome current limitations in treating these metastatic breast cancers. This research develops alendronate conjugated polyethylene glycol functionalized chitosan (ALD-PEG-CHI) NP for the delivery of cell death siRNA (CD-siRNA) and curcumin (CUR) and explores its targeting ability and in vitro cell cytotoxicity. Polyethylene glycol functionalized CHI (mPEG-CHI) NPs serve as control. The size of CD-siRNA loaded NPs is below 100 nm while CUR loaded NPs is below 200 nm, with near neutral zeta potential for all NPs. The CUR encapsulation efficiency (EE) is 70% and 88% for targeted and control NPs, respectively, while complete encapsulation of CD-siRNA is achieved in both NP systems. The bone targeting ability of CY5-dsDNA loaded ALD-PEG-CHI NPs using hydroxyapatite discs is fivefold compared to control indicating ALD presentation at the targeting NP surface. Delivery of CD-siRNA loaded NPs and CUR loaded NPs show synergistic and additive growth inhibition effects against MCF-7 cells by mPEG-CHI and ALD-PEG-CHI NPs, respectively. Overall, these in vitro results illustrate the potential of the targeted NPs as an effective therapeutic system toward bone metastasized breast cancer.

Characterisation of products from EDC-mediated PEG substitution of chitosan allows optimisation of reaction conditions.

PEGylation is a common method use to modify the physiochemical properties and increase the solubility of chitosan (CHI). Knowledge of optimal reaction conditions for PEGylation of CHI underpins its ongoing use in nanomedicine. This study synthesised methoxy polyethyleneglycol grafted CHI (mPEG-CHI) using carbodiimide-mediated coupling. The effect of reagent concentrations and pH on the degree of substitution (DS) and the PEGylation yield (conversion of free PEG to conjugated PEG) was evaluated through detailed chemical characterisation. Within the parameter space investigated, optimised reaction conditions (NH2: COOH:NHS:EDC of 3.5:1:1:10, pH = 5) resulted in a DS of 24 % and a PEGylation yield of 84 %. An EDC-derived adduct formed at pH ≥ 5.5 and at a 15-fold excess of EDC relative to COOH. The adduct was evaluated to be a guanidine derivative formed by the reaction of the amine group of CHI directly with EDC. DS ≥ 12 % imparted water solubility to CHI at physiological pH and mPEG-CHI (0.2-1.0 mg/mL) was not cytotoxic against the breast cancer cell lines MCF-7 and MDA-MB-231, indicating its suitability for medical applications.

Evaluation of the in vivo fate of ultrapure alginate in a BALB/c mouse model.

The linear anionic polysaccharide alginate (ALG) has been comprehensively studied for biomedical applications, yet thus far the in vivo fate of this polymer has not been explored in detail. The current study therefore evaluates the biodistribution of ultrapure ALG (M/G ratio ≥ 0.67 with a measured Mw of 530 kg/mol and polydispersity index; PDI of 1.49) over a 14-day period in BALB/c mice. The biodistribution pattern over 2-days after sample administration using PET imaging with 64Cu-labelled ALG showed liver and spleen uptake. This was confirmed by the 14-day biodistribution profile of cyanine 5-labelled ALG from in vivo and ex vivo fluorescence imaging. Using MacGreen mice confirmed the uptake of the ALG by macrophages in the spleen at the 2-day time point. This extended biodistribution study confirmed the clearance of only a portion of the administered ALG biopolymer, but also uptake by macrophage populations in the spleen over a 14-day period.

Chitosan Nanomedicine in Cancer Therapy: Targeted Delivery and Cellular Uptake.

Nanomedicine has gained much attention for the management and treatment of cancers due to the distinctive physicochemical properties of the drug-loaded particles. Chitosan's cationic nature is attractive for the development of such particles for drug delivery, transfection, and controlled release. The particle properties can be improved by modification of the polymer or the particle themselves. The physicochemical properties of chitosan particles are analyzed in 126 recent studies, which allows to highlight their impact on passive and active targeted drug delivery, cellular uptake, and tumor growth inhibition (TGI). From 2012 to 2019, out of 40 in vivo studies, only 4 studies are found reporting a reduction in tumor size by using chitosan particles while all other studies reported tumor growth inhibition relative to controls. A total of 23 studies are analyzed for cellular uptake including 12 studies reporting cellular uptake mechanisms. Understanding and exploiting the processes involved in targeted delivery, endocytosis, and exocytosis by controlling the physicochemical properties of chitosan particles are important for the development of safe and efficient nanomedicine. It is concluded based on the recent literature available on chitosan particles that combination therapies can play a pivotal role in transformation of chitosan nanomedicine from bench to bedside.

Evaluation of different methods for determination of the iron saturation level in bovine lactoferrin.

Lactoferrin (Lf) samples with ca. 25%, 50%, 75%, 85% and 100% iron saturation were prepared for the purpose of evaluating Chromametry, Differential Scanning Calorimetry (DSC) and Circular Dichroism (CD) spectropolarimetry for their suitability in determining the iron saturation level. Numerical values for colour from Chromametry, enthalpy change of denaturation (ΔHcal) from DSC and molar ellipticities from CD were statistically analysed to evaluate their correlation with the level of iron saturation in Lf. Linear regression analysis of colour coordinates Chroma (C(∗)) and hue (h°) angle on percentage iron saturation level of Lf showed that the values can be used to estimate the iron saturation level. The ΔHcal for the iron saturated peak and the CD ellipticities in the 310-340 nm region provided reliable data for the estimation of iron saturation level of Lf up to 75%. Mono- and di-saturated Lf displayed the same thermal stability and very similar tertiary structures.

Surface chemistry of grafted expanded poly(tetrafluoroethylene) membranes modifies the in vitro proinflammatory response in macrophages.

A series of surface-modified expanded poly(tetrafluoroethylene) membranes showed varied levels of in vitro macrophage proinflammatory response. Membranes containing a mixture of phosphate and hydroxyl groups (as determined by X-ray photoelectron spectroscopy analysis) stimulate greater macrophage activation than samples containing a mixture of phosphate and carboxylic acid segments. The types of proteins that adsorbed irreversibly from serum onto the two samples with the highest and lowest cellular response were investigated using surface-matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Distinct differences in the number and type of proteins that adsorbed were observed between these samples. A correlation was found between the main protein components adsorbed onto the surfaces and the resulting in vitro proinflammatory response. This study strongly supports the hypothesis that the cellular response is not controlled directly by surface properties but is mediated by specific protein adsorption events. This in turn highlights the importance of better understanding and controlling the properties of intelligent surface-modified biomaterials.

Tailorable cell culture platforms from enzymatically cross-linked multifunctional poly(ethylene glycol)-based hydrogels.

As stem-cell-based therapies rapidly advance toward clinical applications, there is a need for cheap, easily manufactured, injectable gels that can be tailored to carry stem cells and impart function to such cells. Herein we describe a process for making hydrogels composed of hydroxyphenyl propionic acid (HPA) conjugated, branched poly(ethylene glycol) (PEG) via an enzyme mediated, oxidative cross-linking method. Functionalization of the branched PEG with HPA at varying degrees of substitution was confirmed via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. The versatility of this hydrogel system was exemplified through variations in the degree of HPA substitution, polymer concentration, and the concentration of cross-linking reagents (horseradish peroxidase and H(2)O(2)), which resulted in a range of mechanical properties and gelation kinetics for these gels. Cross-linking of the PEG-HPA conjugate with a recombinantly produced Fibronectin fragment (Type III domains 7-10) encouraged attachment and spreading of human mesenchymal stem cells (hMSCs) when assessed in both two-dimensional and three-dimensional formats. Interestingly, when encapsulated in both nonfunctionalized and functionalized cross-linked PEG-HPA gels, MSCs showed good viability over all time periods assessed. With tunable gelation kinetics and mechanical properties, these hydrogels provide a flexible in vitro cell culture platform that will likely have significant utility in tissue engineering as an injectable delivery platform for cells to sites of tissue damage.

The in vivo assessment of a novel scaffold containing heparan sulfate for tissue engineering with human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo.

Controlled release of heparin from poly(epsilon-caprolactone) electrospun fibers.

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly(epsilon-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8%w/v PCL in 7:3 dichloromethane:methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury.

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications.

The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of CaP minerals, whereas all the grafted membranes revealed the growth of CaP minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic CaP minerals. Irreversibly adsorbed protein measured after a 1h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro.

Calcium phosphate nucleation on surface-modified PTFE membranes.

Highly porous PTFE membranes are currently being used in facial reconstructive surgery. The present study aims at improving this biomaterial through creating a more bioactive surface by introducing ionic groups onto the surface. The unmodified PTFE membrane does not induce inorganic growth after immersion in simulated body fluid (SBF) for up to 4 weeks. Copolymeric grafting with acrylic acid (AAc) by means of gamma irradiation and subsequent in vitro testing in SBF reveals that this copolymer initially acts as an ion-exchange material and subsequently induces growth of a calcium phosphate phase (Ca/P=2.7) when large amounts (15%) of pAAc are introduced onto the membrane surface. This copolymer is not expected to function well from a biomaterials perspective since SEM showed the pores on the surface to be partly blocked. In contrast, the surface of monoacryloxyethyl phosphate (MAEP)-modified samples is altered at a molecular level only. Yet the modified materials are able to induce calcium phosphate nucleation when the external surface coverage is 44% or above. The initial inorganic growth on these membranes in SBF has a (Ca+Mg)/P ratio of 1.1 (presumably Brushite or Monetite). The secondary growth, possibly calcium-deficient apatite or tricalcium phosphate, has a (Ca+Mg)/P ratio of 1.5. This result is a promising indicator of a bioactive biomaterial.