1. Chitosan Nanoparticles as Drug Carriers
By: Yue Yu

2. Chitosan Nanoparticles (CSNPs) A Promising Drug Delivery System
Stability in nano-scale
Low toxicity
Excellent biocompatibility
Simple and mild preparation method
Versatile routes of administration
Sub-micron size for non-invasive route
Note: The most popular “non-invasive route” is the “mucosal” routes of administration, such as oral, nasal, and ocular mucosa, which will be facilitated by chitosan absorption enhancing effect.
Eur. J. Pharm. Sci. 4: 23-31

3. Conventional Dosage Forms
Why Nano?
Conventional Dosage Forms
Novel Dosage Forms
Only a small amount of administered dose reaches the target site, while the majority of the drug distributes throughout the rest of the body in accordance with its physicochemical and biochemical properties.
Sub
micron DDS
Liposome
Some technical limitations including poor reproducibility and stability, and low drug entrapment efficiency
Polymeric NPs
Better reproducibility and stability profiles, high efficiency to reach the target site, and targeted drug delivery with optimal drug release profiles
Kumar and Banker, 2001

4. Intro –NPs in Pharmaceuitics
Nanoparticles: Solid colloidal particles with diameters ranging from nm. They can be used therapeutically as adjuvant in drug carriers in which the active ingredient is dissolved, entrapped, encapsulated, adsorbed or chemically attached. Polymers used to form NPs can be both synthetic and natural polymers. There are two types of NPs: “nanospheres” and “nanocapsules”. Nanospheres have a monolithic-type structure (matrix) in which drugs are dispersed or adsorbed onto their surfaces. Nanocapsules exhibit a membrane-wall structure and drugs are entrapped in the core or adsorbed onto their exterior.
Naresuan University Journal 2003; 11(3): 51-66

5. Why Chitosan (CS)?
Do you know? Among water-soluble polymers available, CS is one of the most extensively studied. CS is a modified natural carbohydrate prepared by the partial N-deacetylation of chitin, a natural biopolymer derived from crustacean shells such as crabs, shrimps1. It is also found in some microorganisms, yeast and fungi. Because CS possesses some ideal properties of polymeric carriers for nanoparticles (See next slide). It possesses positively charge and exhibits absorption enhancing effect. CSNPs have been extensively developed and explored for pharmaceutical applications2,3.
CS has been widely employed in pharmaceutical and biomedical fields owing to its unique properties such as non-toxicity, biocompatibility, and biodegradability. These characteristics make chitosan an excellent candidate for various biomedical applications such as drug delivery, tissue engineering, and gene delivery.
1. Illum, 1998
2. LeHoux and Grondin, 1993
3. Peniston and Johnson, 1980

7. Nanoparticle delivery systems
Table1. Criteria for ideal polymeric carriers for nanoparticles & nanoparticle delivery systems
Polymeric carriers
Nanoparticle delivery systems
Easy to synthesize and characterize
Simple and inexpensive to manufacture and scale-up
Inexpensive
No heat, high shear forces or organic solvents involved in their preparation process
Biocompatible
Reproducible and stable
Biodegradable
Applicable to a broad category of drugs; small molecules, proteins and polynucleotides
Non-immunogenic
Ability to lyophilize
Non-toxic
Water soluble
Stable after administration
Naresuan University Journal 2003; 11(3)

8. Intro of Preparation method
CSNPs preparation technique has been developed based on chitosan microparticles technology. There are at least four methods available: ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex. The most widely developed methods are ionotropic gelation and self assemble polyelectrolytes. These methods offer many advantages such as simple and mild preparation method without the use of organic solvent or high shear force. In general, the factors found to affect nanoparticles formation including particle size and surface charge are molecular weight and degree of deacetylation of chitosan. The entrapment efficiency is found to be dependent on the pKa and solubility of entrapped drugs. The drug is mostly found to be associated with chitosan via electrostatic interaction, hydrogen bonding, and hydrophobic interaction.
Waree Tiyaboonchai, 2003

9. Applications of CSNPs Non-viral gene delivery vectors
Parenteral administration
Peroral administration
Ocular administration
Delivery of vaccines
Bender et al., 1996; Page-Clisson et al., 1998; Soma et al., 2000
Gerlowski and Jain, 1986; Sadzuka et al., 1998
Norris et al,. 1998; Takeuchi et al., 2001
Pan et al., 2002 ; Vila et al., 2002

10. Folate-CS-DNA NPs for gene therapy
Sania Mansouri, 2006
The mechanism of folic acid (FA) uptake by cells to promote targeting and internalization could improve transfection rates. FA-CS-DNA NPs were prepared using reductive amidation and a complex coacervation process. The aim of the study was to synthesize and characterize FA-CS-DNA NPs and to evaluate their effect on cell viability. The paper shows that FA-NPs have lower cytoxicity, good DNA condensation, positive zeta potential (an abbreviation for electrokinetic potential in colloidal systems) and particle size around 118 nm, which makes them a promising candidate as a non-viral gene vector. Charge ratio (N/P) controlled the nanoparticles size and their zeta potential.

12. Relationship between N/P charge ratio and particle size and zeta potential:
Sania Mansouri, 2006
When the charge ratio of FA-chitosan-DNA nanoparticles is around 1, the nanoparticle size is more than 300nm. If the charge ratio increases, the nanoparticles size decreases to the 120 nm range.

13. With an electrophoresis gel, the plasmid DNA condensation and integrity can be shown:
Sania Mansouri, 2006
This Figure illustrates an intact DNA, before nanoparticle synthesis (lane 1). The DNA in lanes 2 and 3-3a is unable to migrate and remains in the gel loading wells. Following digestion with chitosanase and lysozyme, the plasmid DNA was released from the nanoparticles and could be viewed in lanes 4 and 5-5a.

14. Chitosan nanoparticle as protein/Genes delivery carrier
Methods of preparation
Drugs/Proteins/Genes
CSNPs systems Ionic gelation
Insulin, BSA, cyclosporine A
Coacervation
DNA
Precipitation Emulsion-droplet
Gadopentetic acid
Coalescence Reverse micellar
Doxorubicin
Self-assembly chemical modification
Deoxycholic acid
Quan, 2007

15. Hydrophobically modified glycol chitosan (HGC) as carriers for paclitaxel
Paclitaxel (PTX), an anticancer agent extracted from the bark of the Pacific yew (Taxus brevifolia), has demonstrated significant activity in clinical trials against a variety of tumors. However, PTX is a hydrophobic drug with poor aqueous solubility. To increase its solubility HGC was prepared as a carrier.
HGC conjugates were prepared by chemically linking 5β-cholanic acid to glycol chitosan chains.
In phosphate-buffered saline, the synthesized HGC conjugates formed nano-sized particles with a diameter of 200 nm and exhibited high thermodynamic stability.
Paclitaxel was efficiently loaded into HGC nanoparticles up to 10 wt.% using a dialysis method.
Jong-Ho Kim, 2006

16. The chemical structure of the HGC conjugate is shown below:
HGC nanoparticles show promise as carriers for anticancer peptides and anticancer drugs because they are biocompatible in vivo and accumulate passively in tumor tissue.
Jong-Ho Kim, 2006

17. The average mean diameter of HGC and PTX-HGC NPs were evaluated at 633 nm and 25 oC. The anti-tumor efficacy of PTX-HGC nanoparticles was evaluated in tumor-bearing mice. The survival and the body weight of the mice were recorded. Below is the transmission electron microscopy (TEM) image of HGC alone (left) and PTX-HGC nanoaggregates (right):
PTX was loaded into HGC nanoparticles using a simple dialysis method. The size of the PTX-HGC nanoparticles increased after drug loading from 200 to 400 nm, but the size distribution remained narrow.
Jong-Ho Kim, 2006

18. Deoxycholic acid-conjugated CSNPs (COSDs) for gene carrier
As efficient gene carriers, the COSDs showed superior gene condensation and protection of condensed gene from endonuclease attack.
COSDs showed great potential for gene carrier with the high level of gene transfection efficiencies.
The highly purified COSs, with the average molecular weight of 3000 and 6000 Da, were chemically modified by deoxycholic acid (DOCA).
The synthetic schemes for DOCA conjugation on COSs is shown below:
Su Young Chae, 2005

20. N-trimethyl chitosan chloride (TMC) nanoparticles as protein carriers
TMC nanoparticles were prepared by ionic crosslinking with tripolyphosphate (TPP).
TMC, a chitosan derivative, shows perfect solubility in water over a wide pH range. It also has bioadhesive properties and enhancement of permeability and absorption in neutral and basic-pH environments.
TMC nanoparticles could be prepared by a mild ionic gelation procedure and proved that they are safe carriers for nasal protein delivery.
Two model proteins with similar molecular weight (Mw = 68, 000 Da) but different pI values, bovine serum albumin (BSA, pI = 4.8) and bovine hemoglobin (BHb, pI = 6.8), were to investigate the protein loading and release profiles of TMC nanoparticles.
Fu Chen, 2007

21. TMC precipitation efficacy (PE) can be calculated as follows:
Loading efficiency and loading capacity can be calculated as follows:
Fu Chen, 2007

22. TMC precipitation efficacy as a function of TPP concentration (TMC 2 mg/ml, TPP 0.1–0.9 mg/ml). Data shown are the mean±S.D. (n = 3). TMC33 (▲) and TMC37 (■).
Fu Chen, 2007

23. Fu Chen, 2007
TEM of non-loaded TMC nanoparticles (A), BSA-loaded TMC nanoparticles (B), BHb-loaded TMC nanoparticles (C) and alginate modified nanoparticles (D). (TMC 372mg/ml, TPP 0.6mg/ml, BSA or BHb 0.4mg/ml, sodium alginate 0.3mg/ml). This figure shows the morphology of protein-loaded and nonleaded TMC NPs.

25. Table 1 shows the effect of the initial protein concentration on the LE and LC of the TMC nanoparticles. The high loading efficiency of BSA could be due to the ionic interaction between TMC and BSA. Too high a BSA concentration could lead to aggregation. The influence of the degree of quaternization of TMC on the particle size and zeta potential is shown in Table 2. Nanoparticles prepared by TMC33 showed a larger size and PDI, and a lower zeta potential than those prepared with TMC37 for both loaded and non-loaded formations.
Fu Chen, 2007

26. The figure below compares the permeate efficiency of modified and non-modified TMC nanoparticles, alginate modified TMC nanoparticles at low (5mg/ml) concentration and high (20mg/ml) concentration :
Fu Chen, 2007

27. References:
Characterization of folate-chitosan-DNA nanoparticles for gene therapy Sania Mansouria, Yan Cuieb, Francoise Winnikb
Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system Maryam Amidi, Stefan G. Romeijn
Preparation and evaluation of nanoparticles made of chitosan or N-trimethyl chitosan and a cisplatin–alginate complex S. Cafaggi, E. Russo, R. Stefani
Preparation and modification of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier Yongmei Xu, Yumin Du
Evaluation and modification of N-trimethyl chitosan chloride nanoparticles as protein carriers Fu Chen, Zhi-Rong Zhang
Chitosan nanoparticle as protein delivery carrier - Systematicexamination of fabrication conditions for efficient loading and release Quan Gan, Tao Wang
Chitosan nanoparticles as delivery systems for doxorubicin Kevin A. Janes, Marie P. Fresneau
Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel Jong-Ho Kim, Yoo-Shin Kim, Sungwon Kim
Microencapsulated chitosan nanoparticles for lung protein delivery Ana Grenha, Begona Seijo
In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery Fu Chen, Zhi-Rong Zhang, Fang Yuan