Biosynthesis of gold nanoparticles using marine bacteria and Box–Behnken design optimization

Gold nanoparticles are exciting materials because of their potential applications in optics, electronics, biomedical, and pharmaceutical fields. In recent years, environmentally friendly, low-cost biosynthesis methods with bio-applicable features have continued to be developed for the synthesis of gold nanoparticles. In the present study, an actinobacterial strain was isolated from the Petrosia ficiformis (Poiret 1798) sponge, which was collected from a marine environment, and the gold nanoparticle synthesis was performed for the first time from the bacteria type belonging to the Citricoccus genus. The synthesis conditions were optimized using the Box–Behnken experimental design, with a statistical method that included three independent variables (temperature, time, and mixture ratio) to affect the synthesis at three levels (+1, 0, and ?1). Accordingly, the conditions proposed for the biosynthesis of gold nanoparticles at the maximum optical density values that are specific for the Citricoccus sp. K1D109 strain were estimated as 35°C temperature, 24?h, and 1/5 mixture ratio (cell-free extract/HAuCl₄?·?3H₂O). When recommended conditions were applied, it was determined that the maximum absorbance of the synthesized gold nanoparticles is 1.258 at 545?nm, and their sizes are in the range of 25–65?nm, according to transmission electron microscopy (TEM) data.     

Development,optimization, and in vitro characterization of dasatinib-loaded PEG functionalized chitosan capped gold nanoparticles using Box–Behnken experimental design

Objective: The purpose of this research study was to develop, optimize, and characterize dasatinib loaded polyethylene glycol (PEG) stabilized chitosan capped gold nanoparticles (DSB-PEG-Ch-GNPs).

Methods: Gold (III) chloride hydrate was reduced with chitosan and the resulting nanoparticles were coated with thiol-terminated PEG and loaded with dasatinib (DSB). Plackett–Burman design (PBD) followed by Box–Behnken experimental design (BBD) were employed to optimize the process parameters. Polynomial equations, contour, and 3D response surface plots were generated to relate the factors and responses. The optimized DSB-PEG-Ch-GNPs were characterized by FTIR, XRD, HR-SEM, EDX, TEM, SAED, AFM, DLS, and ZP.

Results: The results of the optimized DSB-PEG-Ch-GNPs showed particle size (PS) of 24.39?±?1.82?nm, apparent drug content (ADC) of 72.06?±?0.86%, and zeta potential (ZP) of ?13.91?±?1.21?mV. The responses observed and the predicted values of the optimized process were found to be close. The shape and surface morphology studies showed that the resulting DSB-PEG-Ch-GNPs were spherical and smooth. The stability and in vitro drug release studies confirmed that the optimized formulation was stable at different conditions of storage and exhibited a sustained drug release of the drug of up to 76% in 48?h and followed Korsmeyer–Peppas release kinetic model.

Conclusions: A process for preparing gold nanoparticles using chitosan, anchoring PEG to the particle surface, and entrapping dasatinib in the chitosan-PEG surface corona was optimized.     

Formulation and optimization of a novel oral fast dissolving film containing drug nanoparticles by Box–Behnken design–response surface methodology

Objective: The purpose of this study was to design and optimize a novel drug nanoparticles-loaded oral fast dissolving film (NP-OFDF) using Box–Behnken design–response surface methodology.

Methods: Drug nanosuspensions produced from high pressure homogenization were transformed into oral fast dissolving film containing drug nanoparticles by casting methods. Herpetrione (HPE), a novel and potent antiviral agent with poor water solubility that was extracted from Herpetospermum caudigerum, was studied as the model drug. The formulations of oral fast dissolving film containing HPE nanoparticles (HPE-NP-OFDF) were optimized by employing Box-Behnken design–response surface methodology and then systematically characterized.

Results: The optimized HPE-NP-OFDF was disintegrated in water within 20?s with reconstituted nanosuspensions particle size of 299.31?nm. Scanning electron microscopy (SEM) images showed that well-dispersed HPE nanoparticles with slight adhesion to each other were exposed on the surface of film or embedded in film. The X-ray diffractogram (XRD) analysis suggested that HPE in the HPE-NP-OFDF was in the amorphous state. In-vitro release study, approximate 77.23% of HPE was released from the HPE-NP-OFDF within 5?min, which was more than eight times compared with that of HPE raw materials (9.57%).

Conclusion: The optimized HPE-NP-OFDF exhibits much faster drug release rates compared to HPE raw material, which indicated that this novel NP-OFDF may provide a potential opportunity for oral delivery of drugs with poor water solubility.     

Development and in vitro–in vivo evaluation of a water-in-oil microemulsion formulation for the oral delivery of troxerutin

Objective: The main objective of this study was to develop and evaluate a W/O microemulsion formulation of troxerutin to improve its oral bioavailability.

Methods: The W/O microemulsion was optimized using a pseudo-ternary phase diagram and evaluated for physical properties. In vitro MDCK cell permeability studies were carried out to evaluate the permeability enhancement effect of microemulsion, and in vivo absorption of troxerutin microemulsion in the intestine was compared with that of solution after single-dose administration (56.7?mg/kg) in male Wistar rats.

Results: The optimal formulation consisted of lecithin, ethanol, isopropyl myristate and water (23.30/11.67/52.45/12.59 w/w) was physicochemical stable and the mean droplet size was about 50.20?nm. In vitro study, the troxerutin-loaded microemulsion showed higher intestinal membrane permeability across MDCK monolayer when compared with the control solution. The W/O microemulsion can significantly promote the intestinal absorption of troxerutin in rats in vivo, and the relative bioavailability of the microemulsion was about 205.55% compared to control solution.

Conclusion: These results suggest that novel W/O microemulsion could be used as an effective formulation for improving the oral bioavailability of troxerutin.     

Box–Behnken design directed optimization for sensitivity assessment of anti-platelet drugs

Optimization of electrospray ionization (ESI) parameters is routinely carried out by one factor at a time (OFAT) or auto-tune software (ATS). Design of experiments (DOE) approach has been reported to be an excellent alternative to OFAT or ATS. Box–Behnken Design (BBD) was successfully used to optimize ESI parameters like nebulizing gas flow rate, desolvation line temperature, heat block temperature, and drying gas flow rate for [M?+?H]⁺ intensity of Clopidogrel bisulfate (CLP) and Ticlopidine (TLP). BBD model was found to be significant with p?<?.0001 for both CLP and TLP. The predicted and optimized (OL) ESI parameters were used for chromatographic analysis and were compared against three levels of ESI parameters, i.e. low level (LL), medium level (ML), and high level (HL). The OL ESI parameters were subjected to chromatographic analysis and its mean peak area was significantly higher than mean peak area for LL, ML, and HL ESI in case of CLP and TLP (p?<?.001). However, no significant difference was observed between the mean peak area for ML and OL of TLP. Thus, BBD can be considered with 29 trials to optimize four mass spectrometric parameters. The liquid chromatographic parameters percentage of methanol, percentage of formic acid and flow rate were also optimized using BBD. However, the optimized method did not significantly influence the peak response over the non-optimized method.     

Design,optimization and evaluation of poly-ɛ-caprolactone (PCL) based polymeric nanoparticles for oral delivery of lopinavir

Lopinavir (LPV)-loaded poly-ε-caprolactone (PCL) nanoparticles (NPs) were prepared by emulsion solvent evaporation technique. Effects of various critical factors in preparation of loaded NPs were investigated. Box–Behnken design (BBD) was employed to optimize particle size and entrapment efficiency (EE) of loaded NPs. Optimized LPV NPs exhibited nanometeric size (195.3?nm) with high EE (93.9%). In vitro drug release study showed bi-phasic sustained release behavior of LPV from NPs. Pharmacokinetic study results in male Wistar rats indicated an increase in oral bioavailability of LPV by 4-folds after incorporation into PCL NPs. From tissue distribution studies, significant accumulation of loaded NPs in tissues like liver and spleen indicated possible involvement of lymphatic route in absorption of NPs. Mechanistic studies using rat everted gut sac model revealed endocytosis as a principal mechanism of NPs uptake. In vitro rat microsomal metabolism studies demonstrated noticeable advantage of LPV NPs by affording metabolic protection to LPV. These studies indicate usefulness of PCL NPs in enhancing oral bioavailability and improving pharmacokinetic profile of LPV.     

Prediction and optimization of nanoclusters-based thermal conductivity of nanofluids: Application of Box–Behnken design (BBD)

The existing models to predict the thermal conductivity of nanofluids are based on single particle diameter, whereas, in actual solutions, nanoparticles mostly exist in a cluster form. Experiments are carried out to observe the effects of various surfactants on stability, nanocluster formation, and thermal conductivity of Al₂O₃–H₂O nanofluid, which is found to be improved considerably with SDS surfactant. The prolonged sonication was not adequate to break the clusters of Al₂O₃ nanoparticles, into an average size of less than 163 nm, indicating the tendency of Al₂O₃ nanoparticles to remain in the form of clusters instead of individual nanoparticles of primary size of 20 nm. Response surface methodology has been employed to design and optimize the experimental strategy by taking volumetric concentration, temperature, and surfactant amount as the contributing factors. The developed model has been validated against the experimental data and the existing models with an accuracy level of ± 8% in the former case. Analysis reveals about the formation of nanoclusters and enhancement in thermal conductivity. The results confirmed that the model can predict thermal conductivity enhancement with an accuracy level of R square value of the order of 0.9766.     

Parametric optimization studies on drilling of sandwich composites using the Box–Behnken design

This study was carried out to investigate the parametric influence on the performance of drilling newly made sandwich composites. Sandwich composite was prepared by using steel and jute as reinforcements and polyester as the matrix material. Drilling experiments were carried out by considering input factors such as spindle speed, feed rate of the spindle, point angle of the drill and tool diameter. Three output factors, namely thrust force developed during drilling, surface roughness of the drilled hole and damage at the entrance surface, were studied. All output factors were optimized by using the Box–Behnken approach, and the best machining conditions were taken on the basis of the desirability approach. Confirmatory experiments were conducted and compared against the Box–Behnken model. The comparison showed only a minor error, and hence the optimization is satisfactory.     

Optimization of methotrexate loaded niosomes by Box–Behnken design: an understanding of solvent effect and formulation variability

Dermal drug delivery system which localizes methotrexate (MTX) in the skin is advantageous in topical treatment of psoriasis. The aim of the current study was to understand dilution effects and formulation variability for the potential formation of niosomes from proniosome gels of MTX. Box–Behnken’s design was employed to prepare a series of MTX proniosome gels of Span 40, cholesterol (Chol-X₁) and Tween 20 (T20-X₂). Short chain alcohols (X₃), namely ethanol (Et), propylene glycol (Pg) and glycerol (G) were evaluated for their dilution effects on proniosomes. The responses investigated were niosomal vesicles size (Y₁), MTX entrapment efficiency percent (EE%-Y₂) and zeta potential (Y₃). MTX loaded niosomes were formed immediately upon hydration of the proniosome gels with the employed solvents. Addition of Pg resulted in a decrease of vesicular size from 534?nm to 420?nm as Chol percentage increased from 10% to 30%, respectively. In addition, increasing the hydrophilicity of the employed solvents was enhancing the resultant zeta potential. On the other hand, using Et in proniosomal gels would abolish Chol action to increase the zeta potential value and hence less stable niosomal dispersion was formed. The optimized formula of MTX loaded niosomes showed vesicle size of 480?nm, high EE% (55%) and zeta potential of –25.5?mV, at Chol and T20 concentrations of 30% and 23.6%, respectively, when G was employed as the solvent. Hence, G was the solvent of choice to prepare MTX proniosomal gels with a maintained stability and highest entrapment.     

In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Development of olmesartan medoxomil optimized nanosuspension using the Box–Behnken design to improve oral bioavailability

The aim of the present investigation was to enhance the oral bioavailability of olmesartan medoxomil by improving its solubility and dissolution rate by preparing nanosuspension (OM-NS), using the Box–Behnken design. In this, four factors were evaluated at three levels. Independent variables include: concentration of drug (X₁), concentration of surfactant (X₂), concentration of polymer (X₃) and number of homogenization cycles (X₄). Based on preliminary studies, the size (Y₁), zeta potential (ZP) (Y₂) and % drug release at 5?min (Y₃) were chosen as dependent responses. OM-NS was prepared by high pressure homogenization method. The size, PDI, ZP, assay, in vitro release and morphology of OM-NS were characterized. Further, the pharmacokinetic (PK) behavior of OM-NS was evaluated in male wistar rats. Statistically optimized OM-NS formulation exhibited mean particle size of 492?nm, ZP of –27.9?mV and 99.29% release in 5?min. OM-NS showed more than four times increase in its solubility than pure OM. DSC and XRD analyses indicated that the drug incorporated into OM-NS was in amorphous form. The morphology of OM-NS was found to be nearly spherical with high dispersity by scanning electron microscopic studies. The PK results showed that OM lyophilized nanosuspension (NS) exhibited improved PK properties compared to coarse powder suspension and marketed tablet powder suspension (TS). Oral bioavailability of lyophilized NS was increased by 2.45 and 2.25 folds when compared to marketed TS and coarse powder suspension, respectively. Results of this study lead to conclusion that NS approach was effective in preparing OM formulations with enhanced dissolution and improved oral bioavailability.     

Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation

Microspheres have been prepared from the resorbable linear polyester of β-hydroxybutyric acid (polyhydroxybutyrate, PHB) by the solvent evaporation technique and investigated in vitro and in vivo. Biocompatibility of the microspheres has been proved in tests in the culture of mouse fibroblast cell line NIH 3T3 and in experiments on intramuscular implantation of the microspheres to Wistar rats for 3 months. Tissue response to the implantation of polymeric microspheres has been found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded. The results of the study suggest that polyhydroxybutyrate is a good candidate for fabricating prolonged-action drugs in the form of microparticles intended for intramuscular injection.     

Application of response surface methodology with a Box–Behnken design for struvite precipitation

Struvite crystals were precipitated by the reaction of magnesium chloride hexahydrate and ammonium dihydrogen phosphate using different concentrations of citric acid as the additive (100, 300, and 500 ppm). The structure, morphology, functional groups and particle size of the crystals were evaluated experimentally by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and particle size analysis. The experimental results demonstrated that citric acid exerted a significant influence on the struvite precipitation and the crystal morphology changed from rod-like to tubular shaped with a larger size and hollow bodies. The average particle size changed from 17.60 to 33.60 μm with increasing citric acid concentration. The results of FTIR suggested that the citric acid adsorbed on the crystal surface. Following the characterization of the crystals prepared using different concentrations of citric acid, the response surface methodology coupled with Box-Behnken design were applied as a statistical tool to determine the effects of the key parameters affecting the precipitation process (temperature, pH and additive concentration) on the responses (namely, particle size and specific cake resistance of struvite). Second-order polynomial equations for both responses were improved to correlate the parameters. Analysis of variance (ANOVA) showed a significant quadratic regression model with high coefficients of the determination values. The optimum conditions for particle size were found to be 60 °C, pH 8 and 500 ppm additive concentration.     

Development and evaluation of a novel drug delivery: Soluplus®/TPGS mixed micelles loaded with piperine in vitro and in vivo

Background: Although piperine can inhibit cells of tumors, the poor water solubility restricted its clinical application. This paper aimed to develop mixed micelles based on Soluplus^® and D-α-tocopherol polyethylene glycol succinate (TPGS) to improve the aqueous solubility and anti-cancer effect.

Methods: Piperine-loaded mixed micelles were prepared using a thin-film hydration method, and their physicochemical properties were characterized. The cellular uptake of the micelles was confirmed by confocal laser scanning microscopy in A549 lung cancer cells and HepG₂ liver cancer cells. In addition, cytotoxicity of the piperine mixed micelles was studied in A549 lung cancer cells and HepG₂ liver cancer cells. Free piperine or piperine-loaded Soluplus^®/TPGS mixed micelles were administered at an equivalent dose of piperine at 3.2?mg/kg via a single intravenous injection in the tail vain for the pharmacokinetic study in vivo.

Results: The diameter of piperine-loaded Soluplus^®/TPGS (4:1) mixed micelles was about 61.9?nm and the zeta potential –1.16?±?1.06?mV with 90.9% of drug encapsulation efficiency and 4.67% of drug-loading efficiency. Differential scanning calorimetry (DSC) studies confirmed that piperine is encapsulated by the Soluplus^®/TPGS. The release results in vitro showed that the piperine-loaded Soluplus^®/TPGS mixed micelles presented sustained release behavior compared to the free piperine. The mixed micelles exhibited better antitumor efficacy compared to free piperine and physical mixture against in A549 and HepG₂ cells by MTT assay. The pharmacokinetic study revealed that the AUC of piperine-loaded mixed micelles was 2.56 times higher than that of piperine and the MRT for piperine-loaded mixed micelles was 1.2-fold higher than piperine (p?Conclusion: The results of the study suggested that the piperine-loaded mixed micelles developed might be a potential nano-drug delivery system for cancer chemotherapy. These results demonstrated that piperine-loaded Soluplus^®/TPGS mixed micelles are an effective strategy to deliver piperine for cancer therapy.     

Central composite rotatable design for optimization of budesonide-loaded cross-linked chitosan–dextran sulfate nanodispersion: characterization,in vitro diffusion and aerodynamic study

Budesonide is a BCS class II drug with low water solubility (0.045?mg/mL) and low oral bioavailability (6–8%) due to high first pass effect. The aim is to prepare cross-linked chitosan–dextran sulfate nanoparticles and/or nanodispersion. Nebulizable cross-linked nanodispersion was prepared by the solvent evaporation technique and characterized through XRPD, FTIR, mean particle size (MPS), polydispersity index (PDI), zeta potential (ZP), drug loading, entrapment efficiency, SEM, % production yield, in vitro diffusion, aerodynamic and stability study. The optimization of formulation was done by using central composite rotatable design to study the effect of independent variables, concentration of chitosan (X1) and concentration dextran sulfate (X2) on the dependent variables, MPS (Y1), drug loading (Y2) and % CDR (% cumulative drug release) (Y3). The MPS, PDI, and ZP of budesonide-loaded nanoparticles were 160.8?±?0.27?nm, 0.36?±?0.04, and 13?±?0.894?mV, respectively. The percent drug loading of all the batches was found in range of 10–16%. The emitted drug in target region (alveoli) was measured by using HPLC and it was found to be 18.26%. It was found that, nanodispersion had the optimum in vitro aerodynamic behavior. Stability study results showed no significant change in MPS, PDI, ZP, and % CDR after three month storage. In conclusion, cross-linked chitosan–dextran sulfate nanoparticles had properties suitable for nebulizable dispersion of increased drug loading, in vitro drug release and avoiding the first pass effect.     

Spray drying formulation of albendazole microspheres by experimental design. In vitro–in vivo studies

Both an experimental design and optimization techniques were carried out for the development of chitosan–pectin–carboxymethylcellulose microspheres to improve the oral absorption of albendazole as a model drug. The effect of three different factors (chitosan, pectin and carboxy methyl cellulose concentrations) was studied on five responses: yield, morphology, dissolution rate at 30 and 60?min, and encapsulation efficiency of the microspheres. During the screening phase, the factors were evaluated in order to identify those which exert a significant effect. Simultaneous multiple response optimizations were then used to find out experimental conditions where the system shows the most adequate results. The optimal conditions were found to be: chitosan concentration, 1.00% w/v, pectin concentration 0.10% w/v and carboxymethylcellulose concentration 0.20% w/v. The bioavailability of the loaded drug in the optimized microspheres was evaluated in Wistar rats which showed an area under curve (AUC) almost 10 times higher than the pure drug.     

Formulation and ex vivo–in vivo evaluation of pH-triggered brimonidine tartrate in situ gel for the glaucoma treatment using application of 32 factorial design

Context: Short residence time, poor bioavailability and poor permeability are the major problems for conventional eye drops treatment.

Objective: The aim of this article is to develop, optimize and ex vivo–in vivo investigation of brimonidine tartrate in situ gel as compared to marketed eye drops for the treatment of glaucoma.

Materials and methods: The effect of independent variables, namely concentrations of polymers, on various dependent variables like viscosity at physiological pH and in vitro drug release were studied by using 3² factorial design. Further the optimized formulation was characterized for ex vivo and in vivo study.

Results and discussion: Experimental data demonstrated that optimized in situ gel formulation (F8) showed in vitro–ex vivo sustained release profile with polymer composites carbopol 974P and HPMC K4M. After 5?h of ex vivo transcorneal permeation study, the amount recovered from the corneal surface on the donor chamber 12.40% (124 ug) and the amount collected from the receptor chamber 76.8% (760 ug) of the initial dose 1?mg. The total amount recovered from the permeation experiment was 89.2%. Bioadhesive carbopol 974P and viscosity HPMC K4M composites optimized formulation (F 8) produce greater influence on the duration of drug action and improved intraocular pressure reduction activity as compared to marketed eye drop solution in in vivo study.

Conclusion: The developed in situ gelling system as a promising ophthalmic formulation to prolong the drug lowering effect on the intraocular pressure.     

Sustained-release of Cyclosporin A pellets: preparation,in vitro release,pharmacokinetic studies and in vitro–in vivo correlation in beagle dogs

The aim of this study was to develop Cyclosporin A (CsA) sustained-release pellets which could maintain CsA blood concentration within the therapeutic window throughout dosing interval and to investigate the in vitro–in vivo correlation (IVIVC) in beagle dogs. The CsA sustained-release pellets (CsA pellets) were prepared by a double coating method and characterized in vitro as well as in vivo. Consequently, the CsA pellets obtained were spherical in shape, with a desirable drug loading (7.18?±?0.17?g/100?g), good stability and showed a sustained-release effect. The C_max, T_max and AUC_0–24 of CsA pellets from the in vivo pharmacokinetics evaluation was 268.22?±?15.99?ng/ml, 6?±?0?h and 3205.00?±?149.55?ng·h/ml, respectively. Compared with Neoral®, CsA pellets significantly prolonged the duration of action, reduced the peak blood concentration and could maintain a relatively high concentration level till 24?h. The relative bioavailability of CsA pellets was 125.68?±?5.37% that of Neoral®. Moreover, there was a good correlation between the in vitro dissolution and in vivo absorption of the pellets. In conclusion, CsA pellets which could ensure a constant systemic blood concentration within the therapeutic window for 24?h were prepared successfully. Meanwhile, this formulation possessed a good IVIVC.     

In vitro evaluation of folic acid modified carboxymethyl chitosan nanoparticles loaded with doxorubicin for targeted delivery

The development of smart targeted nanoparticle that can deliver drugs to direct cancer cells, introduces better efficacy and lower toxicity for treatment. We report the development and characterizations of pH-sensitive carboxymethyl chitosan modified folic acid nanoparticles and manifest their feasibility as an effective targeted drug delivery vehicle. The nanoparticles have been synthesized from carboxymethyl chitosan with covalently bonded bifunctional 2,2′-(ethylenedioxy)-bis-(ethylamine) (EDBE) through the conjugation with folic acid. The conjugation has been analyzed by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The resultant nanoparticles with an average size less then 200 nm measured by dynamic light scattering and transmission electron microscopy. Confocal microscopy and flow cytometric analysis have revealed that folate-mediated targeting significantly enhances the cellular uptake of the nanoparticle and thus facilitates apoptosis of cancer cells (HeLa, B16F1). For the application of the nanoparticles as a drug carrier, Doxorubicin a potent anticancer drug has been loaded into the nanoparticles, with the drug loading amount and the drug release pattern observed.