Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application

In this paper, covalently cross-linked hydrogel composed of N,O-carboxymethyl chitosan and oxidized alginate was developed intending for drug delivery application. In vitro/vivo cytocompatibility and biocompatibility of the developed hydrogel were preliminary evaluated. In vitro cytocompatibility test showed that the developed hydrogel exhibited good cytocompatibility against NH3T3 cells after 3-day incubation. According to the results of acute toxicity test, there was no obvious cytotoxicity for major organs during the period of 21-day intraperitoneal administration. Meanwhile, the developed hydrogel did not induce any cutaneous reaction within 72 h of subcutaneous injection followed by slow degradation and adsorption with the time evolution. Moreover, the extraction of developed hydrogel had nearly 0% of hemolysis ratio, which indicated the good hemocompatibility of hydrogel. Based on the above results, it may be concluded that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel with non-cytotoxicity and good biocompatibility might suitable for the various drug delivery applications.     

pH-responsive polysaccharide-based polyelectrolyte complexes as nanocarriers for lysosomal delivery of therapeutic proteins

Nanopharmaceutics composed of a carrier and a protein have the potential to improve the activity of therapeutical proteins. Therapy for lysosomal diseases is limited by the lack of effective protein delivery systems that allow the controlled release of specific proteins to the lysosomes. Here we address this problem by developing functional polyelectrolyte-based nanoparticles able to promote acidic pH-triggered release of the loaded protein. Trimethyl chitosan (TMC) was synthesized and allowed to form polyelectrolyte complexes (PECs) with the lysosomal enzyme α-GAL through self-assembly and ionotropic gelation, with average particle size <200 nm, polydispersity index (PDI) <0.2, ζ potential of ～ 20 mV, and a protein loading efficiency close to 65%. These polyelectrolyte nanoparticles were stable and active under physiological conditions and able to release the enzyme at acidic pH, as demonstrated by in situ atomic force microscopy (AFM). These nanoparticles were further functionalized with Atto 647N for single-particle characterization and tracking their cellular uptake and fate using high-resolution fluorescence microscopy. In contrast with their precursor, TMC, PECs were efficiently internalized by human endothelial cells and mostly accumulated in lysosomal compartments. The superior physicochemical characteristics of the TMC/α-GAL PECs together with their excellent cellular uptake properties indicate their enormous potential as advanced protein delivery systems for the treatment of lysosomal storage diseases.     

The effect of polyelectrolyte counterion specificity, charge density, and conformation on polyelectrolyte-amphiphile interaction: the carrageenan/furcellaran-amitriptyline system

The effect of polyelectrolyte cation specificity, charge density, and conformation on the interaction between furcelleran, kappa, iota, and lambda-carrageenan, respectively, and amitriptyline, an amphiphilic cationic drug molecule, was studied by means of a dialysis equilibrium technique. The carrageenans used in this study-furcelleran, kappa, iota, and lambda-carrageenan-had a charge density corresponding to 0.69, 0.92, 1.53, and 2.07 sulfate groups per disaccharide. In general, the binding isotherms followed the order Li(+) < Na(+) < N(CH3)(4)(+) < K(+) < or = Cs(+) approximately Rb(+), i.e., the binding isotherms were shifted to higher concentrations of free amphiphile according to the sequence indicated. This affinity sequence correlates well with that found for the dextran sulfate-amitriptyline system (A. Hugerth and L.-O. Sundel?f, Langmuir 2000, 16, 313-317). The factor determining the critical aggregation concentration in the presence of Na(+) compared to K(+) was found to be as follows: the flexibility (conformation) in the case of the lower charged carrageenans, i.e., furcelleran and kappa-carrageenan, charge density for iota-carrageenan, and in the lambda-carrageenan case the difference in the ROSO(3)(-)-alkali ion specificity.     

Hydrodynamic and molecular characteristics of polyelectrolyte complexes between sodium dextransulfate and chitosan hydrochloride

Hydrodynamic and molecular characteristics of particles of polyelectrolyte complexes (PEC) between sodium dextransulfate and chitosan hydrochloride were studied by various physicochemical methods (high-rate sedimentation, viscosimetry, turbidimetry, and diffusion). As was shown, the complex formation is accompanied by increase in the average sizes with simultaneous changes in the shape of the particles of the investigated PEC. According to the proposed polycomplexes model, side by side aggregation of the taken macromolecules could cause disordering of adjacent helical parts of polyanion-matrix that facilitates the formation of sphere-like polycomplexes particles.     

Facile preparation of well-defined near-monodisperse chitosan/sodium alginate polyelectrolyte complex nanoparticles (CS/SAL NPs) via ionotropic gelification: A suitable technique for drug delivery systems

Polymeric nanoparticles have emerged as a promising approach for drug delivery systems. We prepared chitosan (CS)/sodium alginate (SAL) polyelectrolyte complex nanoparticles (CS/SAL NPs) via a simple and mild ionic gelation method by adding a CS solution to a SAL solution, and investigated the effects of molecular weight of the added CS, and the SAL:CS mass ratio on the formation of the polyelectrolyte complex nanoparticles. The well-defined CS/SAL NPs with near-monodisperse particle size of about 160 nm exhibited a pH stable structure, and pH responsive properties with a negatively or positively charged surface. The so-called “electrostatic sponge” structure of the polyelectrolyte complex nanoparticles enhanced their drug-loading capacity towards the differently charged model drug molecules, and favored controlled release. We also found that the drug-loading capacity was influenced by the nature of the drugs and the drug-loading media, while drug release was affected by the solubility of the drugs in the drug-releasing media. The biocompatibility and biodegradability of the polyelectrolytes in the polyelectrolyte complex nanoparticles were maintained by ionic interactions. These results indicate that CS/SAL NPs can represent a useful technique for pH-responsive drug delivery systems.     

In situ gelling of alginate/pluronic solutions for ophthalmic delivery of pilocarpine

We prepared a series of alginate and Pluronic-based solutions as the in situ gelling vehicles for ophthalmic delivery of pilocarpine. The rheological properties, in vitro release as well as in vivo pharmacological response of polymer solutions, including alginate, Pluronic solution, and alginate/Pluronic solution, were evaluated. The optimum concentration of alginate solution for the in situ gel-forming delivery systems was 2% (w/w) and that for Pluronic solution was 14% (w/w). The mixture of 0.1% alginate and 14% Pluronic solutions showed a significant increase in gel strength in the physiological condition; this gel mixture was also found to be free flowing at pH 4.0 and 25 degrees C. Both in vitro release and in vivo pharmacological studies indicated that the alginate/Pluronic solution retained pilocarpine better than the alginate or Pluronic solutions alone. The results demonstrated that the alginate/Pluronic mixture can be used as an in situ gelling vehicle to increase ocular bioavailability.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture

There is significant interest in the development of tissue-engineered skin analogues, which replace both the dermal and the epidermal layer, without the use of animal or human derived products such as collagen or de-epidermalised dermis. In this study, we proposed that alginate hydrogel could be used to encapsulate fibroblasts and that keratinocytes could be cultured on the surface to form a bilayered structure, which could be used to deliver the co-culture to a wound bed, initially providing wound closure and eventually expediting the healing process. Encapsulation of fibroblasts in 2 and 5% w/v alginate hydrogel effectively inhibited their proliferation, whilst maintaining cell viability allowing keratinocytes to grow uninhibited by fibroblast overgrowth to produce a stratified epidermal layer. It was shown that the alginate degradation process was not influenced by the presence of fibroblasts within the hydrogel and that lowering the alginate concentration from 5 to 2% w/v increased the rate of degradation. Fibroblasts released from the scaffold were able to secrete extracellular matrix (ECM) and thus should replace the degrading scaffold with normal ECM following application to the wound site. These findings demonstrate that alginate hydrogel may be an effective delivery vehicle and scaffold for the healing of full-thickness skin wounds.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Preparation and mucoadhesive test of CSA-loaded liposomes with different characteristics for the intestinal lymphatic delivery

Drug delivery to the lymphatic system may be important in terms of the treatment with lymphatic involvement, such as tumor metastases and immunization. Especially, drug transport via the intestinal lymphatics after oral administration has been attracted lots of interests. The purpose of this study was to prepare cyclosporin A (CSA)-loaded liposomes, with different characteristics, and evaluate their ucoadhesivity. Three liposome preparations were formulated: cationic stearylamine liposomes (SA-Lip), anionic phosphatidylserine liposomes (PS-Lip), polymer (chitosan)-coated liposomes (CS-Lip), and characterized. The liposome preparations were found to be spherical in shape, with PS-Lip being the smallest. The liposome preparations exhibited entrapment efficiencies in the order: PS-Lip (52.5±2.9%)>SA-Lip (48.8±3.3%)> CS-Lip (41.7±4.2%). Finally, mucoadhesive tests were carried out using rat intestine, with SA-Lip (67%) showing the best adhesive rate of the three preparations (PS-Lip: 56%, CS-Lip: 61%). These results suggest that a positive charge on the surface of drug carriers may be an important factor for the intestinal drug delivery.     

Biological and polymeric self-assembled hybrid systems: Structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Interaction between SV40 DNA and camptothecin, an antitumor alkaloid

Camptothecin specifically interacted with closed superhelical circular SV40 DNA during incubation in 1.0 M NaCl at 37 degrees C and induced an alkali-labile linkage in the E strand. No interaction occurred in the reaction mixture containing 0.1 M NaCl, or at 4 degrees C. As camptothecin did not affect linear SV40 DNA, the superhelical structure of DNA appeared to be essential. The site of the alkali-labile linkage induced in SV40 DNA I through interaction with camptothecin was near the origin of replication on the basis of the results of experiments with restriction enzymes. Neither sulfhydryl reagents nor EDTA affected the interaction between camptothecin and SV40 DNA I, so the action of camptothecin is different from those of antitumor antibiotics, bleomycin or neocarzinostatin. Analysis of the s20,0w value of SV40 DNA I after the interaction with camptothecin and the sedimentation profiles of DNA after heating in the reaction mixture indicated that the interaction between camptothecin and SV40 DNA I was different from those of intercalating or alkylating agents such as ethidium bromide and methylmethanesulfonate. Replacement of the OH group at C-20 in the E ring of camptothecin by H-, CH3-, and Cl- resulted in the reduction, in this order, of the potency for interaction with SV40 DNA I to induce an alkali-labile linkage.     

Biological and polymeric self-assembled hybrid systems: structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Light scattering,CD, and ligand binding studies of ferrihemoglobin–polyelectrolyte complexes

Quasi‐elastic light scattering (QELS), electrophoretic light scattering (ELS), CD spectroscopy, and azide binding titrations were used to study the complexation at pH 6.8 between ferrihemoglobin and three polyelectrolytes that varied in charge density and sign. Both QELS and ELS show that the structure of the soluble complex formed between ferrihemoglobin and poly(diallyldimethylammonium chloride) [PDADMAC] varies with protein concentration. At fixed 1.0 mg/mL polyelectrolyte concentration, protein addition increases complex size and decreases complex mobility in a tightly correlated manner. At 1.0 mg/mL or greater protein concentration, a stable complex is formed between one polyelectrolyte chain and many protein molecules (i.e., an intra‐polymer complex) with apparent diameter approximately 2.5 times that of the protein‐free polyelectrolyte. Under conditions of excess polyelectrolyte, each of the three ferrihemoglobin–polyelectrolyte solutions exhibits a single diffusion mode in QELS, which indicates that all protein molecules are complexed. CD spectra suggest little or no structural disruption of ferrihemoglobin upon complexation. Azide binding to the ferrihemoglobin–poly(2‐acrylamide‐2‐methylpropanesulfonate) [PAMPS] complex is substantially altered relative to the polyelectrolyte‐free protein, but minimal change is induced by complexation with an AMPS‐based copolymer of reduced linear charge density. The change in azide binding induced by PDADMAC is intermediate between that of PAMPS and its copolymer. © 1999 John Wiley & Sons, Inc. Biopoly 50: 153–161, 1999     

Metal complexes of 1-methyl-3,4-diphenylpyrazole,a ligand formed by the reaction between benzil and N,N,-dimethylhydrazine

New complexes of the general formulae MnX₂L₂ (X = Cl,Br), MnBr₂L₃, CoX₂L₂ (X = Cl, Br, I, NCS, NO₃), NiX₂L₂ (X = Cl, NO₃), NiBr₂L₃·H₂O, NiL₂L₄·H₂O, CuCl₂L, CuBr₂L₂·H₂O, Cu(NO₃)₂L₂, ZnX₂L₂ (X = Cl, Br, NO₃, Zn(NCS)₂L₂·H₂O, CdX₂L₂ (X = I, NO₃) and HgCl₂L, where L is 1-methyl-3,4-diphenylpyrazole, have been prepared and characterized by elemental analysis, conductivity measurements, magnetic moments and spectral (¹H-NMR, IR and electronic) studies. The ligands is formed by the reaction between benzil and N,N-dimethylhydrazine. The nitrogen of the >CN bond is the donor atom to the metal ions. The bis-ligand halide complexes are pseudotetrahedral, while the nitrate complexes contain octahedrally coordinated metal ions. The IR spectra of MCl₂L (M = Cu, Hg) are indicative of the presence of both terminal and bridging metal-halogen bonds supporting polymeric structures. The stereochemistry and the nature of the nickel(II) complexes are markedly dependent upon the anions; the chloride complex is pseudotetrahedral, the iodide square planar, the nitrate polymeric octahedral, while the proposed structural formula for NiBr₂L₃·H₂O comprises Nickel(II) atoms present in both square planar and octahedral coordination environments.     

一种新型蛋白类药物口服载体的海藻酸钠/巯基化果糖-壳聚糖复合水凝胶

壳聚糖经果糖修饰以改善其水溶性,再经半胱氨酸修饰得到巯基化果糖-壳聚糖。海藻酸钠与巯基化果糖-壳聚糖混合溶液经氯化钙及硫酸钠双重交联制得复合水凝胶珠。溶胀试验结果表明:该凝胶珠在pH值6.8及7.4时的溶胀率分别是pH值1.2时的7倍和10倍左右。牛血清白蛋白(BSA)包载试验结果表明:BSA重量为凝胶珠质量的20%时,包载率可达94%以上,随着巯基化果糖-壳聚糖在凝胶珠中比例增加,BSA包载率上升。BSA释放试验表明:pH值1.2时BSA的释放率很低,只有6%～10%的BSA从凝胶珠中释放出来,随后累积释放量基本不变;pH值6.8和pH值7.4时BSA的释放率迅速提高,因此这种复合水凝胶珠可作为一种潜在的口服蛋白类药物载体。     

Diffusion of (de)acylated antibiotic A40926 in alginate and carrageenan beads with or without cells and/or soybean meal

Effective diffusion coefficients (D(e)) of antibiotic A40926 and its deacylated derivative were determined in Ca-alginate (2% wt/wt) and kappa-carrageenan (2.6% wt/wt) gel beads with or without immobilized Actinoplanes teichomyceticus cells and/or soybean meal (SBM). The method used was based on transient concentration changes in a well-stirred antibiotic solution in which gel beads, initially free of solute, were suspended. Unsteady-state diffusion in a sphere was applied and D(e) determined from the best fit of experimental data. A40926 showed markedly different diffusion characteristics than its deacylated derivative. Diffusivity of deacyl-A40926 in alginate or carrageenan gel beads was six to seven times that of A40926. Large differences in partition coefficients (Kp) were also found. In case of beads without additions, A40926, in contrast to deacyl-A40926, strongly partitioned to the liquid phase. Introduction of SBM and/or mycelium in the gel beads decreased the effective diffusivity of deacyl-A40926, but increased its partitioning to the solid phase. Our findings indicate that a relatively moderate structural change of a lipoglycopeptide molecule could lead to a major change in its diffusion/partition characteristics.     

A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.