Synthesis Chitosan Arginine

Chemicals

N-hydroxysuccinimide (NHS)

(130672-5G, 16.60; 130672-25G, 37.40, 130672-100G, 91.30)

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),

 N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride

03450-5G $63.80
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Synthesis

Activate the carboxyl group of L-Arg

Dissolve L-Arg (0.3 g) in distilled water (10 mL). N-hydroxysuccinimide (NHS)

Add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and  
to the solution at a fixed molar ratio of 1:1:3.

Adjust the pH of the resultant solution  to 6 with 1% acetic acid and 1% NaOH solution.

Let react for 2 h.

Conjugate to MW 10 kDa Chitosan 

Dissolve chitosan (1 g, MW 5 kDa)  in 1% acetic acid solution (100 mL).

Adjust pH to 6 as above  with 1% NaOH solution.

Add the activated L-Arg solution to the chitosan solution

React at ambient temperature with continuous stirring for 48 h.

Dialyse the resultant solution  (MWCO = 3,500) against distilled water for 48 h

Lyophilize.

Gao Method

Chitosan  (Mv ˆ 5000, degree of deacetylation ˆ 80%) can be modifed with arginine using EDC and NHS as coupling agents

Dissolve 0.3 g of arginine and 1 g of CS in 50 ml of N, N,N H , N H -tetramethylethylenediamine (TEMED)/HCl buffer solution ( pH 4.7).

Add NHS (0.14 g) and EDC (0.6 g) to this solution

Stir magnetically for 24 h at room temperature.

Dialyse (2000 MWCO) against distilled water for five days.

Lyophilization.

The substitution degree of arginine in CS  is estimated by elemental analysis (C: 45.06%, N: 12.47%, H: 6.83%) to be 20.1%.

Dissolve L-Arg (0.3 g) in distilled water (10 mL). N-hydroxysuccinimide
(130672-5G, 16.60; 130672-25G, 37.40, 130672-100G, 91.30)

(NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
03450-5G
Available to ship on 07/20/14 - FROM 63.80

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bottle 25 G AC15727-0250 200001-334 Each (25g) Direct from Supplier $34.30
bottle 100 g AC15727-1000 200001-336 Each (100g) Direct from Supplier $88.55

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
10 G AC17144-0100 200001-722 Each (10g) Direct from Supplier $86.68
https://us.vwr.com/store/catalog/product.jsp?catalog_number=200001-722

were added to the solution, respectively, at a fixed molar ratio of 1:1:3, to activate the carboxyl group of L-Arg. The pH of the resultant solution was adjusted to 6 with 1% acetic acid and 1% NaOH solution. The carboxyl group of L-Arg was activated for 2 h. Secondly; chitosan (1 g, MW 5 kDa) was dissolved in 1% acetic acid solution (100 mL). The pH was adjusted in the same manner as L-Arg solution with 1% NaOH solution. The activated L-Arg solution was added to the chitosan solution to react at ambient temperature with continuous stirring for 48 h. The resultant solution was dialyzed (MWCO = 3,500) against distilled water for 48 h, then lyophilized. The final product was named as N-ArgCS-A. Using the same method for preparation of N-ArgCS-A, and other chitosans (MW 10 kDa and 20 kDa) NArgCS-B and N-ArgCS-C, were produced, respectively.

Chitosan-Polyethylene Glycol Conjugate

A low molecular weight-chitosan (fw 50000˜150000) was dissolved in a 0.1 M HCl solution at a concentration of 20 mg/ml, and then diluted with a PBS buffer solution (pH 7.4) at a concentration of 2 mg/ml.

5 ml of the prepared chitosan solution (fw 50000˜150000, 10 mg, 6.2×10−5 mol of pyranose units) was mixed with mPEG5k-SC (mPEG5k-succinyl carbonate, fw 5000, 24.84 mg, 4.96×10−6 mol, 8 mol %, IDB, Korea), and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give amPEG5k-LMW-chitosan conjugate (DP 8 mol %; DP: the degree of PEGylation).

Preparation of Polyarginine-Polyethylene Glycol Conjugate
2 ml of 20 mM Tris buffer solution (pH 8.0) was mixed with poly-L-arginine (PLR; fw 15000˜70000, 10 mg, 2.85×10−7 mol, Sigma, USA) and mPEG5k-SC (fw 5000, 7.14 mg, 1.42×10−6 mol), and then subjected to reaction under stirring at room temperature for 2 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a PEG-poly-L-arginine conjugate.

1. Preparation Method of Double Conjugate of Chitosan-Polyamine Polymer Example 1 Preparation of LMW-chitosan-poly-L-arginine (fw 15000˜70000) Conjugate
1-1. A Low molecular weight chitosan (LMW-chitosan; fw 50000˜150000, Fluka, Swiss) was dissolved in a 0.1M HCl solution at a concentration of 20 mg/ml, and then diluted with a 50 mM MES buffer solution (pH 4.0) at a concentration of 2 mg/ml.

1-2. 1 ml of the chitosan solution prepared in Example 1-1 (fw 50000˜150000, 2 mg, 1.24×10−5 mol of pyranose units) was mixed with poly-L-arginine (PLR; fw 15000˜70000, 0.7 mg, 0.2×10−7 mol, Sigma, USA), 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDAC fw 191.7, 383.4 ug, 0.2×10−5 mol), and N-hydroxysulfosuccinimide (Sulfo-NHS; fw 217.14, 434.28 ug, 0.2×10−5 mol), and then subjected to reaction understirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k, Sartorius, Germany), and then freeze-dried to give a LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

1-3. 1 ml of the chitosan solution prepared in Example 1-1 (fw 50000˜150000, 2 mg, 1.24×10−5 mol of pyranose units) was mixed with PLR (fw 15000˜70000, 1.4 mg, 0.4×10−7 mol), EDAC (fw 191.7, 766.8 ug, 0.4×10−5 mol), and Sulfo-NHS (fw 217.14, 869 ug, 0.4×10−5 mol), and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

1-4. 1 ml of the chitosan solution prepared in Example 1-1 (fw 50000˜150000, 2 mg, 1.24×10−5 mol of pyranose units) was mixed with PLR (fw 15000˜70000, 3.5 mg, 1×10−7 mol), EDAC (fw 191.7, 1.917 mg, 1×10−5 mol), and Sulfo-NHS (fw 217.14, 2.17 mg, 1×10−5 mol), and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

2. Preparation Method of Triple Conjugate of Polyethylene Glycol-Chitosan-Polyarginine Example 13 Preparation of mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) Conjugate
13-1. A LMW-chitosan (fw 50000˜150000) was dissolved in 0.1M HCl solution at a concentration of 20 mg/ml, and then diluted with aPBS buffer solution (pH7.4) at a concentration of 2 mg/ml.

13-2. 10% mPEG2k-LMW-chitosan-poly-L-arginine (PLR ) (fw 15000-70000) conjugate

10 ml of the chitosan solution prepared in Example 13-1 (fw 50000-450000, 20 mg, 1.24×10−4 mol of pyranose units) was mixed with mPEG2k-SG (mPEG2k-succimidyl glutarate, fw 2000, 24.8 mg, 1.24×10−5 mol, 10 mol %, IDB, Korea), and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG2k-LMW-chitosan (fw 50000˜150000) conjugate (DP 10 mol %; DP: the degree of PEGylation). 2 mg of the mPEG2k-LMW-chitosan (fw 50000˜150000) conjugate was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with (fw 15000˜70000, PLR 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol). The mixture was subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate (DP 10 mol %).

13-3. 15% mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate

10 ml of the chitosan solution prepared in Example 13-1 (fw 50000˜150000, 20 mg, 1.24×10−4 mol of pyranose units) was mixed with mPEG2k-SG (fw 2000, 37.26 mg, 1.863×10−5 mol, 15 mol %), and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG2k-LMW-chitosan (fw 50000˜150000) conjugate (DP 15 mol %). 2 mg of the mPEG2k-LMW-chitosan (fw 50000˜150000) conjugate was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol). The mixture was subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate (DP 15 mol %).

25-1. 5% mPEG5k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate

10 ml of the chitosan solution prepared in Example 13-1 (fw 50000˜150000, 20 mg, 1.24×10−4 mol of pyranose units) and mPEG5k-SC (fw 5000, 31.05 mg, 6.21×10−6 mol, 5 mol %, IDB, Korea) were mixed together, and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG5k-LMW-chitosan (fw 50000˜150000) conjugate (DP 5 mol %). 2 mg of the prepared mPEG5k-LMW-chitosan (fw 50000˜150000) conjugate was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 386.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG5k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate (DP 5 mol %).

25-2. 15% mPEG5k-LMW-chitosan-poly-L-arginine (fw 15000-70000) conjugate

10 ml of the chitosan solution prepared in Example 13-1 (fw 50000˜150000, 20 mg, 1.24×10−4 mol of pyranose units) and mPEG5k-SC (fw 5000, 46.58 mg, 9.316×10−6 mol, 15 mol %) were mixed together, and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG5k-LMW-chitosan (fw 50000˜150000) conjugate (DP 15 mol %). 2 mg of the prepared mPEG5k-LMW-chitosan (fw 50000˜150000) conjugate was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 386.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug. 0.4×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a mPEG5k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate (DP 15 mol %).

7-1. Preparation of mPEG2K—COCH(CH3)Cl

mPEG2K—OH (0.5 g, 0.25 mmol), 4-dimethylaminopyridine (DMAP; 91.6 mg, 0.75 mmol), and triethylamine (TEA; 0.070 ml, 0.5 mmol) were dissolved in 12 ml of dichloromethane (DCM), and then slowly added to a solution of 2-chloropropionyl chloride (0.12 ml, 1.25 mmol) dissolved in 3 ml of DCM at 0° C. under nitrogen gas, and then the reaction temperature was raised from 0° C. to room temperature, followed by stirring for 24 hrs. The reaction was confirmed by TLC. The reaction mixture was concentrated under reduced pressure, and precipitated in diethyl ether, filtered, and purified. The resultant was recrystallized in ethanol, filtered, and purified. The structure of the compound was confirmed by 500 MHz 1H-NMR.

37-2. Preparation of Phthaloyl LMW Chitosan (phth-LMW-chitosan)

A LMW-chitosan (LMW-Chitosan Fluka, 500 mg, 3.1 mmol) and phthalic anhydride (Phthalic anhydride 1.38 g, 9.32 mmol) were dissolved in 10 ml of dimethylformamide (DMF), and the solution was heated to 130° C. under nitrogen gas, followed by stirring for 7 hrs. The reactant was cooled to room temperature, and the precipitate was washed with ice water, and filtered. The resulting solid was purified with ethanol by soxhlet extraction. The resulting solid was dried under vacuum at 50° C. to give a phth-LMW-chitosan having a protected amine group of chitosan.

37-3. Preparation of 15 mol % PEGylated 5′-mPEG2K-LMW-chitosan

A phth-LMW-chitosan (70 mg, 0.24 mmol) prepared in Example 37-2 was dissolved in 1 ml of pyridine, and the solution mixed with a solution of mPEG2K—COCH(CH3)Cl (72.2 mg, 36.1 μmol) prepared in Example 37-1 in 2 ml of DMF. The mixture was heated to 100° C. under nitrogen gas, followed by stirring for 24 hrs. The reactant was cooled to room temperature, and precipitated in ethanol. The resultant was washed with ethanol and ether, and filtered. The resulting solid 5′-mPEG2K-chitosan (phth), in which 5′C hydroxy group of chitosan was conjugated with PEG, was dried under vacuum. 50 mg of 5′-mPEG2K-chitosan (phth) was dissolved in 10 ml of hydrazine monohydrate (H2NNH2.H2O), and the solution was refluxed under nitrogen gas at 130° C., while stirring for 24 hrs. The reactant was cooled to room temperature. The precipitate was purified, and mixed with 10 ml of 0.1 M NaOH (aq), followed by stirring at room temperature for 16 hrs. The pH of the mixture was adjusted to 10 with 50 mM HCl (aq) by dialysis, and freeze-dried to give a compound 5′-mPEG2K-LMW-chitosan. The compound was confirmed by 500 MHz 1H-NMR.

37-5. 15% 5′-mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000)

2 mg of 5′-mPEG2K-LMW-chitosan prepared in Example 37-3 was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a 5′-mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

37-6 10% 5′-mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000)

2 mg of 5′-mPEG2K-LMW-chitosan prepared in Example 37-4 was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a 5′-mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

49-4. 15% 5′-mPEG5k-LMW-chitosan-poly-L-arginine (fw 15000˜70000)

2 mg of 5′-mPEG5K-LMW-chitosan prepared in Example 49-2 was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a 5′-mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

49-5. 10% 5′-mPEG5k-LMW-chitosan-poly-L-arginine (fw 15000˜70000)

2 mg of 5′-mPEG5K-LMW-chitosan prepared in Example 49-3 was dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), and Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k), and then freeze-dried to give a 5′-mPEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate.

61-1. Preparation of Man-PEG2K—NHS

PEG2K-diamine (0.5 g, 25 μmol) was dissolved in 25 ml of DCM, and Fmoc-OSu (12.6 mg, 37.5 μmol) was added thereto at 0° C., followed by TLC. The stirring was performed to exhaust PEG-diamine. A vacuum distillation apparatus was used to remove DCM. The resultant was dissolved in a small amount of DCM, precipitated in ether, and purified to give Fmoc-PEG-NH2 containing (Fmoc)2-PEG. The PEG-compound, Fmoc-PEG-NH2 was monitored by TLC. The prepared Fmoc-PEG-NH2 (0.46 g, 23 μmol) was dissolved in 23 ml of DCM. Succinic anhydride (23.0 mg, 230 μmol) and pyridine (22.3 μl, 276 μmol) were added thereto, and refluxed under stirring and nitrogen gas at 40-60° C. overnight. The reactant was cooled to room temperature, extracted with DCM, and washed with saturated NH4Cl(aq). Water was removed from the DCM layer with MgSO4, and the vacuum distillation apparatus was used to remove DCM. The resultant was dissolved in a small amount of DCM, precipitated in an excessive amount of ether, and purified to give Fmoc-PEG-COOH containing (Fmoc)2-PEG. The Fmoc-PEG-COOH compound was monitored by TLC, and then dissolved in D2O, followed by 500 MHz 1H NMR. The Fmoc-PEG-COOH compound (0.45 g, 22.5 μmol) was dissolved in 50% piperidine/DMF, and stirred for 1.5 hrs for Fmoc deprotection. The reactant was precipitated in an excessive amount of ether, washed with ether, filtered, and purified to give H2N-PEG-COOH containing PEG-diamine. The obtained compound H2N-PEG-COOH (0.4 g, 20 μmol), mannose (10.8 mg, 60 μmol), and imidazole (8.2 mg, 120 μmol) were dissolved in 2 ml of N-methylpyrrolidone (NMP), heated to 60° C., and stirred for 20 hrs. The reactant was cooled to room temperature, extracted with DCM, and washed with saturated NH4Cl(aq). Water was removed from the DCM layer with MgSO4, and the vacuum distillation apparatus was used to remove DCM. The resultant was purified using a silica gel column chromatography. PEG was recovered using DCM/MeOH/NH4OH, and the solvent was removed using the vacuum distillation apparatus. The resultant was dissolved in a small amount of DCM, precipitated in an excessive amount of ether, and purified to give Man-PEG-COOH containing (Man)2-PEG. The Man-PEG-NHS compound was subjected to NMR, so as to confirm the mannose.

A solution of DCC (12.9 mg, 62.5 μmol) in 0.3 ml of DCM was added dropwise to a solution of the Man-PEG-COOH compound (125 mg, 6.25 μmol) and NHS (7.2 mg, 62.5 μmol) dissolved in 1.5 ml of DCM under nitrogen gas below 0° C. for 1 hr. The mixture was stirred for 10-12 hrs while maintaining the low temperature. Filtering was performed to remove the resulting solid (ureas), and precipitated in ether. Recrystallization was performed from EA (ethyl acetate), and purified to give Man-PEG-NHS. The Man-PEG-NHS compound was dissolved in D2O for the confirmation of mannose and dissolved in CDCl3 for the confirmation of NHS, and then subjected to 500 MHz 1H NMR.

61-2. 15% Man-PEG2k-LMW-chitosan-poly-L-arginine (fw 15000-70000) conjugate

10 ml of the chitosan solution (fw 50000˜150000, 20 mg, 1.24×10−4 mol of pyranose units) prepared in Example 13-1 and Man-PEG2K—NHS (fw 2000, 37.26 mg, 1.863×10−5 mol, 15 mol %) prepared in Example 61-1 were mixed, and then subjected to reaction under stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k) and freeze-dried to give a Man-PEG2K-LMW-chitosan (fw 50000˜150000) conjugate (DP 15 mol %). 2 mg of the prepared Man-PEG2K-LMW-chitosan (fw 50000˜150000) conjugate were dissolved in 1 ml of 50 mM MES buffer solution (pH 4.0), and then mixed with PLR (fw 15000˜70000, 0.7 mg, 0.2×10−7 mol), EDAC (fw 191.7, 383.4 ug, 0.2×10−5 mol), Sulfo-NHS (fw 217.14, 434.28 ug, 0.2×10−5 mol), followed by stirring at room temperature for 24 hrs. The mixture was purified using a centrifugal filter (MWCO 30k) and then freeze-dried to give a Man-PEG2k-LMW-chitosan-poly-L-arginine (fw 15000˜70000) conjugate (DP 15 mol %).

Protocol 2

Poly-l-arginine hydrochloride (PLR) (molecular weight = 13,300 Da): Sigma

1,2-distearoyl-sn-glycero-3-phos-phoethanolamine--[methoxy(polyethylene glycol)-2000] (DSPE-PEG): Avanti Polar Lipids

The synthesis procedure of PLR–PEG was based on the pre-
viously described method (Huang et al., 2002). Briefly, PLR was
dissolved in 25 ml of 50 mM sodium tetraborate buffer (STBB, pH
8.5) per gram of PLR. The resulting solution was stirred vigor-
ously for approximately 30 min and subsequently filtered through
a 0.22 ␮m Durapore ® membrane (Sterile Millex GV, Sigma–Aldrich,
Buchs, Switzerland) into a sterile culture tube. The appropriate stoi-
chiometric amount of DSPE-PEG powder was then slowly added to
the solution while it was continuously stirred. After another 6 h
of vigorous stirring at room temperature, the solution was trans-
ferred to a dialysis tube (Spectr/Por dialysis tubing, M.W.C.O. of
6–8 kDa, Spectrum Laboratories, Inc., Rancho Dominguez, CA). The
synthesized product was dialyzed out for 24 h in 10 mM phosphate-
buffered saline (PBS, pH 7.0), followed by an additional 24 h of
dialysis in deionized water. The product was then freeze-dried for
48 h at −70 ◦ C with a pressure of 0.2 mbar.

The synthesis schemes of CS–PLR and PEG–CS–PLR conjugates are
summarized in Suppl. Fig. 1. PLR was covalently linked to CS via
formation of amide bonds between the amine groups (–NH 2 ) of CS
and the carboxyl groups (–COOH) of PLR. The formation of amide
bonds between the activated carboxyl groups (–COOH) of PLR and
amine groups (–NH 2 ) of CS resulted in the conjugates of CS–PLR. The
conjugation of PLR to CS and the pegylation of CS–PLR conjugates
were confirmed by 1 H NMR (data not shown). The grafting of PLR and
PEG to CS was tested by FI-IR spectra. PEG–CS–PLR showed the
characteristic absorption band around 2900 cm − 1 and 1650 cm − 1 ,
resulting from the increased C–H bond and amide (C O) bond for-
mation by grafting of PEG to CS–PLR.

Preparation  of Octaarginine R8 -CS

Octaarginine (R8) was synthesized by Fmoc (9-fluor-enylmethyloxy-carbonyl)-solid-phase peptide on a Rink amide resin (Futaki et al. 2001). It was conjugated to chitosan via carbodiimide chemistry, as shown in Scheme 1 (Liu et al. 2004). Briefly, the carboxyl group of R8 was activated by EDC/NHS dissolved in MES buffer (0.1 M MES, pH 6.0) at room
temperature for 10 h, with 1:5:2 molar ratio of octaarginine:EDC:NHS. The amino end of R8 was protected by acetyl. Chitosan was then added and allowed to react for another 24 h by stirring. The reaction was quenched by adding hydroxylamine to a final concentration of 10 mM, and the pH value was brought to 8.0 with NaOH. The resulting solution was dialyzed by using Spectra/Pro membrane (MWCO = 12,000–14,000) against distilled water for 4 days before lyophilization. Herein, two conjugation degrees of R 8 -CS were prepared with molar ratios of R8 to chitosan residues as 1:8, 1:4, which were denoted as R 8 -CS1 and R 8 -CS2, respectively.