Applied Biochemistry and Biotechnology

, Volume 169, Issue 1, pp 239–249

Preparation of Hyaluronic Acid Micro-Hydrogel by Biotin–Avidin-Specific Bonding for Doxorubicin-Targeted Delivery


  • Yuan Cui
    • School of Materials Science and EngineeringChangchun University of Science and Technology
  • Yanhui Li
    • School of Materials Science and EngineeringChangchun University of Science and Technology
    • School of Materials Science and EngineeringChangchun University of Science and Technology
  • Toyoji Kakuchi
    • Division of Biotechnology and Macromolecular Chemistry, Graduate school of EngineeringHokkaido University

DOI: 10.1007/s12010-012-9968-1

Cite this article as:
Cui, Y., Li, Y., Duan, Q. et al. Appl Biochem Biotechnol (2013) 169: 239. doi:10.1007/s12010-012-9968-1


Hyaluronic acid is a naturally ionic polysaccharide with cancer cell selectivity. It is an ideal candidate material for delivery of anticancer agents. In this study, hyaluronic acid (HA) micro-hydrogel loaded with anticancer drugs was prepared by the biotin–avidin system approach. Firstly, carboxyl groups on HA were changed into amino groups with adipic acid dihydrazide (ADH) to graft with biotin by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride named as HA–biotin. When HA–biotin solution mixed with doxorubicin hydrochloride (DOX·HCl) was blended with neutravidin, the micro-hydrogels would be formed with DOX loading. If excess biotin was added into the microgel, it would be disjointed, and DOX will be released quickly. The results of the synthesis procedure were characterized by 1H-NMR and FTIR; ADH and biotin have been demonstrated to graft on the HA molecule. A field emission scanning electron microscope was used to observe morphologies of HA micro-hydrogels. Furthermore, the in vitro DOX release results revealed that the release behaviors can be adjusted by adding biotin. Therefore, the HA micro-hydrogel can deliver anticancer drugs efficiently, and the rate of release can be controlled by biotin-specific bonding with the neutravidin. Consequently, the micro-hydrogel will perform the promising property of switching in the specific site in cancer therapy.


Hyaluronic acidBiotin–avidinMicro-hydrogelDrug deliveryDOX


Hyaluronic acid is a naturally ionic polysaccharide, the major component of the extracellular matrix. It has attracted much attention in recent years due to its potential applications in the vitreous, synovial fluid, and cancer metastasis and plays pivotal roles in wound healing, cell differentiation, and motility [1, 2]. Hyaluronic acid (HA) has the ability of cancer cell selectivity because two kinds of HA receptors: CD44 and RHAMM are overexpressed in cancer cells. CD44 is a family of glycoproteins originally associated with lymphocyte activation; RHAMM is the receptor for HA-mediated cell motility [3, 4]. The overexpression of HA-binding receptors on cancer cells leads to the enhancing of the cancer selectivity of HA. Hence, HA is a very attractive material for delivery of anticancer agents such as anticancer drugs by conjugation to drug molecules, grafting hydrophobic or cationic side chains, as well as cross-linking of HA chains into microgels [5, 6].

Wooram Park et al. [7] selected different degrees of acetylated hyaluronic acid with low molecular weight (AC-HALM) to form self-organized nanogels to load the anticancer drug doxorubicin (DOX). For the work, physicochemical characteristics examinations and in vitro test showed the DOX-loaded AC-HALM nanogel was a good candidate for development as an effective anticancer drug carrier. Hyun-Jong Cho et al. [8] synthesized polyethylene glycol (PEG)-conjugated hyaluronic acid-ceramide (HACE) to prepare DOX-loaded HACE-PEG-based nanoparticles. In vitro DOX release from HACE-based nanoparticles was sufficiently sustained, and efficient cellular uptake of DOX via HA and CD44 interaction was also exhibited in the CLSM study. As a result, PEGylated HACE nanoparticles represent a promising anticancer drug delivery system for cancer diagnosis and therapy. Awesh Kumar Yadav et al. [9] synthesized hyaluronic acid-poly(ethylene glycol)-poly(lactide-co-glycolide) (HA-PEG-PLGA) copolymer to make nanoparticles with DOX loading. They found DOX-loaded HA-PEG-PLGA nanoparticles were able to deliver a higher amount of DOX as compared with mPEG-PLGA nanoparticles. DOX-loaded surface-modified nanoparticles are able to deliver DOX within the tumor by receptor-mediated endocytosis and the enhanced permeability and retention effect.

Hyaluronic acid is also a fully biocompatible candidate for modification with adipic acid dihydrazide to form hydrogels or change carboxyl groups into amino groups for further modification [1012]. In this work, the biotin–avidin system is introduced in HA to cross-link the HA molecule into the micro-hydrogel with DOX loading to perform the function of a switch.

The biotin–avidin system (BAS) is a rapid developmental technique in biological sciences. Avidin is a tetrameric biotin-binding protein produced from the oviducts of birds, reptiles, and amphibians and deposited in the whites of their eggs. Avidin can be linked to four molecules of biotin simultaneously with a high degree of affinity and specificity. Biotin–avidin-specific binding has been used to obtain a hydrogel or grafting and cross-linking in some previous researches [13, 14]. Neil MacKinnon et al. [15] optimized lipid vesicle binding to polymeric hydrogel micro-beads via the avidin–biotin conjugation system and characterized the stability of the resulting microgel-bound liposomes. Jongseong Kim et al. [16, 17] investigated bio-responsive hydrogel micro-lenses with stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgels. These hydrogel micro-particles were then functionalized with biotin to form cross-links by multivalent binding of avidin to biotin on the hydrogel micro-lenses. Jason D. Clapper et al. [18] described a novel biotinylated nano-templated degradable hydrogel that could be rapidly outwardly engineered under mild aqueous conditions using the biotin–avidin interaction. Neutravidin is a deglycosylated version of avidin. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity.

Herein, the biotin–avidin system approach was used to obtain the HA micro-hydrogel with DOX loading. HA was modified by adipic acid dihydrazide (ADH) to let the biotin conjugate with the HA molecule resulting in HA–biotin. HA has been widely investigated as a target in anticancer drug delivery because it binds with the CD44 receptor; in the process, DOX will be mixed with the HA–biotin solution to obtain a DOX-loaded carrier. Finally, neutravidin was blended with the HA–biotin–DOX solution to give HA micro-hydrogel with DOX loading. If excess biotin was added into the micro-hydrogel, the gel would be disjointed to perform the properties of switching. Then, DOX will be released from the micro-hydrogel rapidly. The HA microgel based on biotin–neutravidin bonding is highly promising for the targeted delivery of anticancer drugs.

Materials and Methods


Hyaluronic acid sodium salt (HA, Mn = 1.5 to 1.8 MDa) and neutravidin were purchased from Fluka (Czech Rep.) and Sigma (St. Louis, MO, USA), respectively. Biotin and ADH were purchased from Alfa Aesar. Doxorubicin hydrochloride (DOX·HCl) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. N,N′-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), and N-(3-dimethyl-propyl)-N-ethylcarbodiimidde hydrochloride (EDC·HCl) were purchased from GL Biochem (Shanghai) Ltd.. All other reagents were purchased from Beijing Chemical Works unless otherwise noted.

Synthesis of HA–ADH and HA–ADH–HA Hydrogel

HA sodium salt (100 mg) and ADH (435.4 mg) were mixed and dissolved in 18 mL water with magnetic stirring. Then, 2 mL EDC·HCl solution (15 mg/mL) was added in the mixture at pH 4.9 that was adjusted by dropping HCl. After stirring at room temperature for 2 h, the solution was dialyzed against deionized water for 24 h. The solution was freeze-dried, and then, the HA–ADH floccule was obtained. In addition, the HA–ADH–HA hydrogel was prepared by modulating the dosage of the cross-linking agent and EDC.

Synthesis of HA–Biotin

Biotin (4.1 mmol), NHS (4.1 mmol), and DCC (4.5 mmol) were dissolved in 50 mL N,N-dimethylformamide and reacted at 50 °C for 16 h with magnetic stirring. Then, white precipitate was filtrated by a Buchner funnel. The filtrate was added into cold ether gradually until no precipitate appeared; the precipitate was collected as biotin–NHS (as shown in Fig. 1.).
Fig. 1

Preparation of biotin–NHS

HA–ADH (88 mg) and biotin–NHS (4 mg) were dissolved in 20 mL PBS solution (pH = 7.4) and reacted at room temperature for 24 h with magnetic stirring to give HA–biotin [19]. The product was introduced into a dialysis tube (molecular weight cutoff (MWCO) of 10,000) to dialyze against deionized water for 24 h. The product was freeze-dried and HA–biotin was obtained.

Preparation of HA Micro-Hydrogel with DOX Loading

Five milliliters of HA–biotin (10 mg/mL) and 0.8 mg DOX·HCl mixture were blended with 0.6 mL neutravidin (1 mg/mL) dropped dropwisely under stirring to form HA micro-hydrogel loaded with DOX·HCl. The HA/DOX micro-hydrogel was then transferred to the dialysis tubing (MWCO, 20,000 Da) and dialyzed against deionized water for 24 h to remove any free polymer or drug and lyophilized to obtain the DOX-loaded microgels. The reaction procedures of each step of gel formation were shown in Fig. 2. The drug loading content and efficiency of the DOX-loaded HA micro-hydrogel were calculated with the following equations:
Fig. 2

The carboxyl groups on HA modified with ADH to react with biotin–NHS. Neutravidin was introduced to bond with biotin–HA to get micro-hydrogel

$$ Drug\,loading\,content=\frac{{actual\,amount\,of\,DOX\,\,in\,gel}}{{amount\,of DOX-loaded\,gel}}\times 100\% $$
$$ Drug\,loading\,efficiency=\frac{{actual\,amount\,of\,DOX\,\,in\,gel}}{{input\,amount\,of\,DOX\,\,in\,gel}}\times 100\% $$

In vitro DOX·HCl release behaviors from the DOX-loaded HA micro-hydrogel were investigated in phosphate-buffered saline (PBS) at pH 7.4. Two milliliters of the DOX-loaded HA micro-hydrogel solution (1 mg/mL) was transferred in 10 mL of PBS to a dialysis tube (MWCO, 3,500 Da). The release medium was stirred at 100 rpm at 37 °C. At predetermined intervals, 2 mL of PBS was taken out and an equal volume of fresh PBS was replenished. The amount of released drug was determined by UV spectrophotometry at 480 nm.


Fourier transform infrared spectra (FTIR) of HA, HA–ADH, and HA–biotin were obtained by a Bruker Vertex 70 infrared electro-photometer using the potassium bromide (KBr) method. Spectra represent an average of 64 scans with removed CO2 peaks. 1H-NMR spectra were recorded in D2O at 25 °C on a Unity-400 NMR spectrometer.

The morphologies of the HA–ADH–HA hydrogel and HA–biotin–neutravidin–HA micro-hydrogel were visualized by FSEM. HA hydrogels were imaged following critical point drying (Polaron, UK) to remove water. The cross section, the surface adjacent to the Teflon dish, and the surface at the air interface were visualized. The dehydrated hydrogels were coated with gold and imaged with a Micrion FEI Philips Model XL 30 SEM at 20 kV.

Results and Discussions

Critical Condition of HA–ADH–HA Hydrogel Formation

Because ADH had the capability to cross-link HA into hydrogel, in order to get HA–ADH solution rather than HA–ADH–HA hydrogel, critical condition of hydrogel formation was necessary. The critical condition was obtained by modulating solution pH and dosage of ADH and EDC. The state of the hydrogel will change from liquid to soft fluid or brittle solid. The results were listed in Table 1.
Table 1

Critical condition of HA–ADH–HA hydrogel formation



pH (adjusted with HCl)



Fluid hydrogel

2.5 mg/mL

3.5 ≤ pH ≤ 4.7

0.1 mol equiv.

0.5 mol equiv.

Solid hydrogel

8 mg/mL

pH ≤ 3.5

1.5 mol equiv.

1.0 mol equiv.

As shown in Table 1, the amount of EDC and ADH, pH of the solution, and the concentration of HA will influence the product from HA–ADH solution to HA–ADH–HA hydrogel. During the hydrogel formation, the pH of the solution was the most critical condition. When the other reaction conditions were fixed, the solution had a tendency to form a hydrogel when the pH was smaller than 4.7. As a result, in order to obtain HA–ADH solution, the condition was controlled as follows: HA sodium salt (100 mg) and ADH (435.4 mg) were dissolved in 18 mL water, and then 2 mL EDC (15 mg/mL) was added in the mixture, and the pH of the reaction solution was adjusted to 4.9 with HCl.

1H-NMR of HA–ADH and HA–Biotin

In the process of HA–ADH preparation, the amount of EDC was demonstrated to be a critical factor influencing the grafting percentage of ADH which was calculated by 1H-NMR. The 1H-NMR spectrum of HA–ADH in D2O was shown in Fig. 3. The degree of modification was determined by comparison of the integration of the hydrazide group in ADH (2.2–2.4 ppm) (peak Ha in Fig. 3) with that of the acetamido moiety of the N-acetyl-d-glucosamine residue of HA (1.9 ppm) (peak Hb in Fig. 3), which indicated that ADH had been conjugated to HA. As shown in the spectrum, 28.04 % grafting percentage of ADH is obtained [20].
Fig. 3

1H-NMR spectrum of HA–ADH

Although the carboxyl group on biotin could react with the amino group and hydroxy group, the product of carboxylic acid reacting with amino was ammonium salt but not carboxamide. As a result, the application will be hindered. When the carboxyl group was activated by NHS, it is easy to graft onto HA–ADH.

The 1H-NMR spectrum of HA–biotin in D2O was shown in Fig. 4. It can be seen clearly in Fig. 4 that peaks a (1.6 ppm) and b (4.6 ppm) correspond to H of –CH2CH2CO and –CH(CH)NH in biotin as shown in the spectrum, respectively. That indicated the biotin had been grafted on HA–ADH successfully.
Fig. 4

1H-NMR spectrum of HA–biotin

FTIR Spectrum

In this study, the FTIR spectrum (Fig. 5) was used to confirm the dihydrazide reaction and biotin grafting. As can be seen in the spectrum, C=O stretching of glucuronic acid at 1607 cm−1 of HA disappeared. There was a newly formed peak at 1,558 and 1,556 cm−1associated with the N–H function group of HA–ADH and HA–biotin compared with HA. And the peak at 1,646 cm-1 was seen to appear in HA–biotin corresponding to the amide I band of the acetamido group. At the same time, compared with the IR curve of HA–ADH, the amide absorption peaks of HA–biotin shifted from 1,613 and 1,558 cm−1 to 1,603 and 1,556 cm−1, respectively, corresponding to the consumption of the hydrazide group to form a bond between HA–ADH and biotin [21, 22].
Fig. 5

FTIR spectra of HA, HA–ADH, and HA–biotin

And it also can be seen in Fig. 5 that the characteristic peaks changed no more. The result indicated that in the procedure of grafting, the chains of hyaluronic acid remain.

Micro-Hydrogel Formation and DOX Loading

DOX·HCl is an anthracycline anticancer drug which is widely used to treat various solid malignant tumors. Cancers treated with doxorubicin include: bladder, breast, head and neck, leukemia (some types), liver, lung, lymphomas, mesothelioma, multiple myeloma, neuroblastoma, ovary, pancreas, prostate, sarcomas, stomach, testis (germ cell), thyroid, and uterus [2326]. For this reason, DOX was used as the model drug capsuled in HA micro-hydrogel in this study.

In the study, the neutravidin was used as cross-linker to link HA–biotin into the microgel with the specific binding of biotin and neutravidin. In this process, DOX resolved in HA–biotin solution will be loaded in the microgel. If excess biotin was added into the micro-hydrogel, they will compete with neutravidin that has bound to HA–biotin resulting in micro-hydrogel disjointing. It was a desirable property of micro-hydrogel with reversible switching. Formation and disjointing of HA micro-hydrogel are shown in Fig. 6. Then, the loaded drug will release at the specific site rapidly from the microgel.
Fig. 6

Procedures of HA–biotin–neutravidin–HA micro-hydrogel formation and DOX loading and release. I Synthesis of HA–biotin, II neutravidin is introduced in HA–biotin to from micro-hydrogel with DOX·HCl loading, III excess biotin is added to disjoint the micro-hydrogel resulting in rapid release of the loaded drug

Morphologies of HA and Hydrogels

SEM morphologies of the HA–ADH–HA hydrogel and HA–biotin–neutravidin–HA micro-hydrogel revealed differences in their microstructures. The surface of the HA–ADH–HA hydrogel had pores (Fig. 7a) and tended to form lamellas. The surface layer was uniform and continuous, while the image of the HA–biotin–neutravidin–HA (Fig. 7b) microgel revealed high subtle porosity throughout the surface, with comparative uniform pores ranging in diameter from 5 to 10 mm. Furthermore, the micro-hydrogel had a subtle three-dimensional structure grid where some micro-beads dispersed in the mesh. Due to the special structure, the endued ability of the HA micro-hydrogel can maintain drug to perform drug delivery.
Fig. 7

Morphologies of freeze-dried HA–ADH–HA (a) and HA–biotin–neutravidin–HA (b)

In Vitro DOX·HCl Loading and Release

In this study, the drug release behaviors of the HA micro-hydrogel were investigated with PBS and biotin PBS solution. The release rate can be controlled by biotin adding.

DOX·HCl as a hydrophilic anticancer drug was loaded into the HA micro-hydrogel vesicle in the process of gel formation. The drug loading content and drug loading efficiency were 3.8 and 52.6 %, respectively. The in vitro DOX·HCl release behaviors were investigated at pH 7.4 in PBS.

The DOX-loaded HA micro-hydrogel was dispersed in a dialysis tube containing PBS buffer. The in vitro release of DOX was examined and results were displayed in Fig. 8.
Fig. 8

DOX released from the HA micro-hydrogel, dialyzed against PBS, and compared with the PBS–biotin solution

As shown in Fig. 8, the DOX release rate was accelerated by the presence of biotin, and over 90 % loaded DOX was released in 40 h. Therefore, only about 60 % of the initial loading amount was released from the HA micro-hydrogel at pH 7.4 without biotin in PBS. As a result, the HA micro-hydrogel loaded with anticancer drugs will perform the promising property of targeted cancer therapy as well as specific site switching


HA has the ability of cancer cell selectivity because two kinds of HA receptors: CD44 and RHAMM are overexpressed in cancer cells. So, HA was selected in this study to load DOX to form the drug delivery vehicle with cancer cell target. A specific binding by the BAS approach for HA micro-hydrogel formation and DOX·HCl loading and release was performed in this paper. Hyaluronic acid with ADH was synthesized to use as the reactant for further grafting with biotin. In the process, the amount of ADH and EDC, the concentration of HA, and pH of the solution were critical reaction conditions that should be controlled to obtain the HA–ADH solution rather than the HA–ADH–HA hydrogel. Therefore, carboxyl groups on HA were modified into amino groups to react with biotin–NHS. In brief, water-soluble HA bearing lateral chains endowed with biotin mixed with DOX·HCl blended with neutravidin, and the DOX-loaded micro-hydrogel was formed. The drug loading content and drug loading efficiency were 3.8 and 52.6 %, respectively. The results of 1H-NMR and FTIR demonstrated that the procedure of ADH and biotin grafting was successful, and grafting percentage of ADH on HA was 28.04 % calculated by 1H-NMR. FESEM was used to observe micromorphologies of HA and hydrogels that indicated the microstructure of the HA–biotin–neutravidin–HA micro-hydrogel exhibits higher subtle porosity with micro-beads. When excess biotin was added into the drug-loaded HA micro-hydrogel, it will be disjoined to release the drug rapidly. Then, the HA micro-hydrogel loaded with anticancer drugs will perform the promising property of switching in the specific site in targeted cancer therapy.


The authors are thankful to the National Natural Science Foundation of China (50903009), Jilin science & technology department, science and technology development project (20100115, 20070566) and Science & Technology Bureau of Changchun City project (08SF58) for financial support to this work.

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© Springer Science+Business Media New York 2012