Cross-linked hyaluronic acid matrix and film experiments with specific plasmid DNAs

Summary

Natural polysaccharides are being extensively studied as carriers for gene delivery. The non-inflammatory and non-immunogenic nature of hyaluronic acid (H A ) is particularly important from the point of view of clinical application. In addition, the presence of hyaluronidase in the body promotes the degradation of hyaluronic acid carriers. The encapsulation of D N A into cross-linked hyaluronan materials degrades the hyaluronan in the presence of the enzyme, thereby releasing the DNA-carrying HA fragments. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".

Operation method

Preparation of HA-DNA substrates and films

Move

Preparation of HA-DNA substrates and films Materials

reagents

Diacetyl Hexanedioate (ADH; Sigma-Aldrich)

A nti-F lag antibody (Sigma-A ldrich).

C0 S--1 cells (A T C C , R ockville, M aryland).

Dimethylformamide (Sigma-Aldrich)

Dimethylmethoxymethylamine (Sigma-Aldrich) DMEM culture base (C ellgro, Heindorn, Virginia)

Ethyl-3-[3-dimethylammonio]propylcarbodiimide (ED C I) (Sigma-Aldrich)

Fetal Bovine Serum (FB S) (C ellgro)

FGM-2. B ulletK its Basis (R oche D iagnostics)

H A Quantification Kit (C orgenix, W e stm in ste r, Colorado)

H oescht 33342 Photographic Dye (e x : 485n m , e m : 530n m ; M olecular P ro b es).

H A ( K raeber G m bH & Co., W a ld h o fstr, G erm an y ) storage liquid (10m g/m l, i. e. ,1% )

Transparentease (S g m a -A ldrich)

Hydrochloric acid, lm o l/ L H C l

Isopropyl alcohol (80 %, 90 % and 100 %; F ischer C hemical)

MC 285 Rabbit anti-mouse HAS 2 polyclonal antibody (from D r. John McDonald, University of Utah) and peroxidase-labeled sheep anti-rabbit secondary antibody.

Normal neonatal human skin fibroblasts (NHDF; C lonetics).

Phosphate Buffer (PBS, Sigma-Aldrich)

Plasmid D N A (p D N A, various concentrations), dissolved in sterile water

pFlag-C M V -5a Mammalian Expression Vector

This vector has c D N A encoding the P D G F or H A S 2 cloning site.

P D G F Test Kit Q uantikine E L IS A K it (R & D S ystem s)

Instrumentation

Aluminum weighing pan (sterilized, 4 mm diameter) (V W R S cien tific )

C ytofluorom eter (C ytofluor-I I , B iosearch, B edford, M assach u setts)

37°C incubator (VWR Scientific)

Freeze dryer (Freezem obile 12E L, G ard in er, N ew York)
Swinging bed (L abQ uake shaker L-1237, Lab In d u strie s, B erkeley, C alifornia)

Methods

Preparation of HA-DNA Substrates and Films

1- Slowly mix I m l of a suitable p D N A solution with 20 m l of I % H A solution.

2. Transfer 8 ml of the above H A /p D N A mixture to each aluminum tray. Then proceed as follows.

H A - D N A substrate

a . Suddenly freeze and lyophilize the HA-DNA mixture to form a solid HA-DNA matrix. b .

b - Dissolve IOOmg of A D H in a IOOml mixture of 9 0 % D M F /10% H 2O . Incubate the above solid HA-DNA substrate with this A D H solution for 30 m i n .

c- Dissolve 120 mg of EDCI in the mixture from the previous step and initiate the cross-linking reaction by adjusting the p H to 5 with lm o l/L of H C l .

d-Pour off the solution, pour in IOO ml of 90% isopropanol, and repeatedly wash the HA-DNA substrate with 90% isopropanol, and finally with 100% isopropanol.

e. Evacuate the HADNA matrix. The dried material is shown in Figure IA (Kimet al. 2003).

HA-DNA film

a-Air-dried HA/pDNA mixture to form HA-DNA film.

b. Dissolve I O m g A D H in I O Oml 8 0 % isopropanol and partially rehydrate the dried film by soaking it in this solution for 3 0 m i n .

c. Add 1 ml of EDCI solution (12 mg/ml in water) to the above mixture and start the crosslinking reaction by adjusting the p H to 5 with 1 mol/L of H C l .

d. Remove the solution, add 100 ml of 90% isopropanol and wash the film repeatedly with 90% isopropanol and finally with 100% isopropanol.

e. Air dry the HADNA film. The dried film material is shown in Figure IIB (Kim et al. 2003).

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