A Novel Targeting Drug Delivery System Based on Self-Assembled Peptide Hydrogel

doi:10.4236/jbnb.2011.225074

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Journal of Biomaterials and Nanobiotechnology, 2011, 2, 622-625

doi:10.4236/jbnb.2011.225074 Published Online December 2011 (http://www.scirp.org/journal/jbnb)

A Novel Targeting Drug Delivery System Based on

Self-Assembled Peptide Hydrogel

Liang Liang1*, Jun Yang1, Qinghua Li1, Ming Huo1, Fagang Jiang2, Xiaoding Xu3,

Xianzheng Zhang3

1Department of Ophthalmology, The First College of Clinical Medical Science, China Three Gorges University, Yichang, China;

2Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,

China; 3Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan,

China.

E-mail: *[email protected]

Received September 12th, 2011; revised October 24th, 2011; accepted November 25th, 2011.

ABSTRACT

In the last two decades, 5-fluorouracil (5-FU) is widely used in clinica l practice to inhibit the fibrob lasts to proliferate

and improve the success rate of glaucoma-filtering surgery, but 5-FU has many toxic effects to normal ocular tissues.

The self-assembled peptide hydrogels may serve as a new class of biomaterials for applications including tissue engi-

neering and drug delivery. How to deliver 5-FU quickly and precisely to the target sites of ocular tissue by a self-as-

sembled peptide hydrogel remains unexplored. RGD (arginine-glycine-aspartic acid) sequence is cell attachment site in

extracellular matrix (ECM). Thus, If the self-assembled peptide hydrogel containing the RGD sequence that act as a

specific attachment site for the pro liferated fibroblasts adhesion could be d esigned, after integrated 5-FU, a novel tar-

geting drug delivery system will be put into practice in the future.

Keywords: Dr ug Delivery System, Self-Assembly, Filtering Surgery

1. Introduction

The success rate of glaucoma-filtering surgery unfortu-

nately has been limited by postoperative scarring [1].

Scar formation results from infiltration of fibroblasts into

damaged areas, proliferation of those fibroblasts, and

synthesis of ECM glycoproteins. For treatment of scar

formation, 5-FU was widely reported as adjunct to im-

prove surgical results by inhibiting the postoperative pro-

liferation of fibroblasts of the filtering site [2-4]. Yet, It

was reported toxic effects of 5-FU included toxicity on

the conjunctival and corneal epithelium, wound dehis-

cence, and wound leaks [2], some of which are vision

threatening [3]. If a drug delivery system could be de-

signed to specifically target the proliferated fibroblasts

after the filtering surgery, not only the success rate of

surgery will be significantly improved, but also the pos-

sible toxic effects of 5-FU to the surrounding normal

ocular tissues will be avoided eventually.

A family of peptides has been developed whose ability

to self-assemble into supramolecular hydrogel material is

directly linked to their intramolecularly folded state.

These peptides adopt random coil conformations in aque-

ous solution and are freely soluble until intramolecular

folding is triggered by the addition of a stimulus. Upon

folding, the peptides adopt a conformation conducive to

self-assembly. Assembly ultimately leads to the forma-

tion of a structurally hydrogel without the need for in-

corporation of covalent crosslinks [5]. This self-assem-

bled peptide hydrogels may serve as a new class of bio-

materials for applications including tissue engineering

and drug delivery system [6-9]. Given that the degrada-

tion products consist of the drug and amino acid, this

drug delivery system has an advantage over polymer-

based drug delivery system that generate polymer frag-

ments with heterogeneous chain lengths upon degrada-

tion that may present complex toxicity profiles [10].

The RGD sequence was discovered as cell attachment

site in ECM some 20 years ago [11], and the receptors

for these RGD sequence were identified and organized in

the integrin family. The integrin family is one of the most

important cell adhesion molecules which are found on

the cell surface that act as receptors for cell-to-cell and

cell- ECM adhesion [12-14].

Whether can we design a novel targeting drug delivery

A Novel Targeting Drug Delivery System Based on Self-Assembled Peptide Hydrogel623

system based on self-assembled peptide hydrogel which

contains the RGD peptide sequence that act as a specific

receptor for the proliferated fibroblasts adhesion?

We hypothesized that a novel peptide containing a

bioactive RGD sequence was designed and prepared.

When dissolving the peptide in distilled water, a su-

pramolecular hydrogel with nanofibers was formed

through the self-assembly of the peptide. In addition, this

self-assembled peptide hydrogel could integrate 5-FU

into its nanofibers during the process of self-assembly of

the peptide. The proliferated fibroblasts can be attached

in the peptide hydrogel through the recognition of the

RGD sequence, and 5-FU is delivered to the fibroblasts

by this model of targeting of drugs (Figure 1).

In the past decade, hydrogels formed from self-as-

sembled proteins or peptides have attracted considerable

attention. Unlike the conventional polymeric hydrogels

that are made of covalently crosslinked polymers, pro-

tein- or peptide- based hydrogel is composed of peptide

molecules that self-assemble from aqueous solution into

cylindrical nanofibers that display bioactive epitopes on

their surfaces [15]. The bioactive epitopes could be rec-

ognized by some receptors on cell surface, allowing cell

adhesion to self-assembled peptide hydrogel [14]. In ad-

dition, this self-assembled peptide hydrogel forms a net-

work of nanofibers that are similar in scale to the natural

ECM and therefore provides an “in vivo” environment

for cell growth, migration, and differentiation [5]. Actu-

ally, much recent effort has been focused on the synthe-

sis of short peptides to create a new generation of

self-assembled materials for the use in biomedical appli-

cations. One of the well studied short peptides is the

build block comprising of a specific peptide sequence

Figure 1. The novel model of targeting of drug based on

peptide hydrogel with nanofibers.

and a hydrophobic aromatic tail such as the popular

N-Fluorenyl-9-methoxycarbonyl (FMOC) group, which

has an ability to self-assemble into hydrogel by taking

advantage of π-π stacking interactions [16,17].

Based on this point, a novel peptide containing a bio-

active RGD sequence and FMOC tail could be synthe-

sized and the corresponding hydrogel formed subse-

quently. The specific experiments include the peptide

synthesis and peptide hydrogels preparation.

The peptide was synthesized manually in 1.98 mmol

scale on the 2-chlorotrityl chloride resin employing a

standard FMOC solid phase peptide synthesis (SPPS)

method. Before the reaction, the resin was washed with

CH2Cl2 (three times) and DMF (three times) and then

immersed in DMF for 30 min. After draining off DMF

solution, a DMF solution of the mixture of FMOC pro-

tected amino acid (4 equiv relative to resin loading) and

DiEA (6 equiv) was added to the resin and shaken for 2 h

at room temperature. After removing the reaction solu-

tion, the resin was washed with DMF (three times). Sub-

sequently, 20% piperdine/DMF (V/V) solution was in-

troduced to the resin to remove the FMOC protected

groups. After shaking for 30 min at room temperature,

the reaction solution was drained off and the resin was

washed with DMF (three times). The presence of free

amino groups was indicated by a blue color in the Kaiser

test. Thereafter, a DMF solution of the mixture of FMOC

protected amino acid (4 equiv), HBTU (4 equiv), HOBt

(4 equiv) and DiEA (6 equiv) was added. After shaking

for 1.5 h at room temperature, the reaction solution was

drained off and the resin was washed with DMF (three

times). The absence of free amino groups was indicated

by a yellow color in the Kaiser test. After repetition of

the deprotection and acylation reaction, the resin was

finally washed with DMF (three times) and CH2Cl2

(three times) and dried under vacuum for 24 h. Cleavage

of the expected peptide and the removal the protected

groups of side chains from the dried resin were per-

formed using a mixture of TFA, deionized water, and

TIS in the ratio of 95:2.5:2.5. After 2 h shaking at room

temperature, the cleavage mixture and three subsequent

TFA washing were collected. The combined solution was

concentrated to a viscous solution by rotary evaporation.

Cold ether was added to precipitate the product. After

washing with cold ether (five times) to remove TFA re-

sidual, the precipitate was dissolved in distilled water and

then freeze-dried under vacuum for 3 days. The obtained

crude products were purified by high-pressure liquid

chromatography (HPLC) with a C18 column and using a

linear gradient of acetonitrile and DI water containing

0.1% TFA. IR: ~3470 cm amide A band, ~1657 cm−1

amide I band, ~1558 cm−1 amide II band; ESI-MS:

1009.4, found: 1008.4 (M-H)-.

A Novel Targeting Drug Delivery System Based on Self-Assembled Peptide Hydrogel

624

After peptide synthesis, peptide was well dissolved in

ultra purified water to form 1.5 wt% peptide solution and

subsequently filtrated for the sterilization. After placing

at room temperature for 30 min, a well defined peptide

hydrogel appeared based on the self-assembly of the pep-

tide molecules.

Simultaneously, it is possible to design this kind of

novel peptide containing a bioactive RGD sequence to

specifically bind the fibroblasts due to the expression of

high level of 1 integrin in proliferated fibroblastsɑ [18].

Because hydrogels have been widely applied as intelli-

gent carriers in controlled drug delivery systems [19-21],

5-FU could be integrated into this self-assembled peptide

hydrogel and released upon enzyme-mediated hydrogel

degradation [10].

Currently, filtration surgery has been recognized as a

standard therapy for glaucoma, which involves generat-

ing a filtration fistula to allow the escape of aqueous

humor to reduce the intraocular pressure (IOP). The suc-

cess rate of glaucoma filtration surgery is limited by the

postoperative scarring formation. The scarring formation

is usually attributed to the proliferation of fibroblasts at

the surgical site after glaucoma filtering surgery, leading

to the scleral flap fibrosis and eventual filtration failure.

In the case of evaluation of effect of this novel targeting

drug delivery system in filtering surgery of rabbit eye,

we chose the clinical observation and pathology analysis

that could test the decrease amount of intraocular pres-

sure, the duration of bleb, the present of filtration fistula

and the proliferated status of fibroblasts in surgical site.

After administrating this self-assembled peptide hy-

drogel containing 5-FU in filtering surgery intraopera-

tively, the success rate of surgery will be improved by a

localized and targeting delivery of a very small yet effi-

cient amount of the antifibroblastic agent 5-FU, offering

potential benefits to decrease the toxic side effects in

patients who are at high risk of failed filtering surgery.

2. Conflicts of In t e r e st S t a t e m e nt

The authors have no conflict of interests.

3. Acknowledgements

Financial support was obtained from National Natural

Science Foundation of China (30772382, 50633020) and

National Key Basic Research Program of China

(2005CB623903).

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