DOI: 10.1002/chem.201202038

Bioresponsive Hyaluronic Acid-Capped Mesoporous Silica Nanoparticles forTargeted Drug Delivery

Zhaowei Chen,[a, b] Zhenhua Li,[a, b] Youhui Lin,[a, b] Meili Yin,[a] Jinsong Ren,*[a] andXiaogang Qu*[a]

Introduction

The major goal of chemotherapy is to ensure the safety andefficient transportation of drug molecules to the targetedsites. Over the past two decades, advances in biotechnologyand nanotechnology have brought about a paradigm shift todesign more sophisticated approaches for drug deliveries.Recently, mesoporous silica nanoparticles have attracted at-tention as promising compounds of multimodal nanoparticlesystems owing to their straightforward synthesis, tunablepore sizes, large load capacity, good chemical stability, andexcellent biocompatibility. These interesting features havemade these scaffolds ideal for the design of nanodevices and“on-command” delivery applications.[1] To date, severalmeso ACHTUNGTRENNUNGporous silica nanoparticles (MSP)-based controlled-re-lease systems have been synthesized by using different kindsof capping agents including nucleic acids,[2] organic mole-cules,[3] inorganic nanoparticles,[4] polymers,[5] and supramo-lecular assemblies.[1a, 6] The porous nanovectors could re-

spond to specific external triggers, such as redox,[3c,d, 7]

pH,[2b, e, 8] temperature,[2a,5a] enzymes,[2a,9] competitive bind-ing,[6] and photoirradiation.[3a,b, 10] Although promising, manyof the existing capping agents have disadvantages, such aspoor applicability in aqueous solutions and biocompatibility,and the toxicity of the capping agents used. Thus, to developoptimal platforms for anticancer therapeutic applications,the following major issues of capping agents must be consid-ered: 1) biocompatibility—the toxicity of the cap is a majorconcern for potential applications; 2) targeting ability—thespecial affinity between the cap and cancer cells can en-hance the intracellular uptake of nanoparticles, thereby im-proving therapeutic efficiency; 3) selectivity—disintegrationof the cap autonomously inside cells of interest can facilitateefficient delivery of anticancer drugs and reduce side ef-fects; 4) stability—colloidal stability improves blood circula-tion lifetimes, which are crucial for potential in vivo drugdelivery; and 5) chemical simplicity—the development of asimple and robust technique not only minimizes the leak ofencapsulated cargoes over the design and construction pro-ACHTUNGTRENNUNGcess but also is important for future use in practical applica-tions. Therefore, the search for effective caps that, in partic-ular, combine end-capped properties and bioactive targetingability simultaneously still remains a big challenge in thisfield.

Herein we address these issues by developing MSP-basednanoreservoirs that are end-capped with hyaluronic acid(HA) and demonstrate great potential for cell-specific tar-geted and controlled drug release in response to specific en-zymes present at tumor sites. As one of the extracellularmatrix components, HA is a negatively charged, naturallyoccurring polysaccharide with nonimmunogenic, biocompat-

Abstract: In this paper, we present afacile strategy to synthesize hyaluronicacid (HA) conjugated mesoporoussilica nanoparticles (MSP) for targetedenzyme responsive drug delivery, inwhich the anchored HA polysaccha-ACHTUNGTRENNUNGrides not only act as capping agents butalso as targeting ligands without theneed of additional modification. Thenanoconjugates possess many attractivefeatures including chemical simplicity,high colloidal stability, good biocom-

patibility, cell-targeting ability, and pre-cise cargo release, making them prom-ising agents for biomedical applica-tions. As a proof-of-concept demon-stration, the nanoconjugates are shownto release cargoes from the interiorpores of MSPs upon HA degradation

in response to hyaluronidase-1 (Hyal-1). Moreover, after receptor-mediatedendocytosis into cancer cells, the anch-ored HA was degraded into small frag-ments, facilitating the release of drugsto kill the cancer cells. Overall, we en-vision that this system might open thedoor to a new generation of carriersystem for site-selective, controlled-re-lease delivery of anticancer drugs.

Keywords: cancer therapy · drugdelivery · enzymes · mesoporousmaterials · nanoparticles

[a] Z. Chen, Z. Li, Y. Lin, M. Yin, Prof. Dr. J. Ren, Prof. Dr. X. QuState Key Laboratory of Rare Earth Resource Utilizationand Laboratory of Chemical BiologyChangchun Institute of Applied ChemistryChinese Academy of Sciences, ChangchunJilin 130022 (P.R. China)E-mail : jren@ciac.jl.cn

xqu@ciac.jl.cn

[b] Z. Chen, Z. Li, Y. LinGraduate school of Chinese Academy of SciencesBeijing, 100039 (P.R. China)

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201202038.

� 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2013, 19, 1778 – 17831778

ible, and biodegradable characters.[11] Intriguingly, it hasbeen used as a drug carrier or targeting moiety due to thespecific interaction with CD44.[11,12] In addition to its princi-pal receptor CD44, HA also interacts with several cell sur-face receptors, including RHAMM (receptor for hyaluron-an-mediated motility, CD168 involving in wound healingand cancer), HARE (HA receptor for endocytosis), andToll-like receptors-2 and -4 (recognizing hyaluronan oligo-ACHTUNGTRENNUNGmers in dendritic cells during inflammatory events).[11d]

CD44 is overexpressed in many solid tumor cells and previ-ous studies have demonstrated that CD44 is a major recep-tor for HA.[13] Also, HA could be readily degraded by lyso-somal enzyme hyaluronidase-1 (Hyal-1), which is the majorenzyme found in the tumor microenvironment, into low mo-lecular weight fragments after being endocytosed by cancercells.[11,14] We then seek to take advantage of these uniquefeatures to explore the possibility of using HA as cappingagents for targeted, enzyme-responsive drug delivery appli-cations.

Results and Discussion

As shown in Scheme 1, HA macromolecules were anchoredto the openings of the MSPs through 1-ethyl-3-(3-dimethyl-ACHTUNGTRENNUNGaminopropyl)carbodiimide (EDC) reaction and were utiliz-

ed as caps to encapsulate the guest molecules within theporous channels. Specific enzymatic hydrolysis of the HAshell would open the nanopores, permitting the selective re-lease of previously entrapped cargoes. To the best of ourknowledge, this is the first study in which the HA polymeris employed as both capping and targeting agent. As aproof-of-principle experiment, rhodamine B was chosen asmodel molecule and Hyal-1 was utilized for HA degrada-tion. The opening of the capped system was tested by meas-uring the enzymatic triggered dye release from the MSPs.Importantly, we have successfully demonstrated the target-ing ability of the nanoconjugates toward CD44 overex-pressed cancer cells and efficient intracellular-controlleddrug delivery.

The mesoporous silica nanoparticles were prepared by fol-lowing a base-catalyzed sol–gel procedure,[15] and the result-ing porous silica particles (~100 nm in diameter) that con-tained hexagonally arranged pores were characterized byTEM, SEM, and X-ray diffraction (Figure 1A and Fig-

ACHTUNGTRENNUNGures S1 and S2 in the Supporting Information, respectively).The N2 adsorption–desorption isotherms of the particlesshowed a typical type IV curve with a specific surface areaof 1280 m2 g�1, an average pore diameter of 2.9 nm, and anarrow pore distribution (see Figure S3 in the SupportingInformation). Then, the surfaces of the resulting MSPs weremodified by 3-(2-aminoethylamino)propyltrimethoxysilaneto obtain amino-functionalized mesoporous silica nanoparti-cles (designed as MSNs). The resulting MSNs were allowedto be capped with HA through EDC/N-hydroxysuccinimide(NHS) chemistry (designed as MSN-HA). Importantly, theelectrostatic interaction between the remaining amino andcarboxyl groups facilitated HA efficiently and evenly dis-tributed around the surfaces of MSNs, which could increasethe nanoconjugates� colloidal stability and inhibit the molec-ular diffusion from the pores. The successful coating processof MSPs was validated by various spectroscopy methods.TEM images (Figure 1B) provided direct evidence of theHA distribution on the amino-functionalized MSP material.Although no clear difference in shape and average diameterbetween native MSNs and capped MSNs was observed (Fig-ure 1A and B), the hardly detectable mesoporous structureand the clear border of the treated MSNs indicated that HAmolecules were efficiently immobilized on the surfaces. Fur-thermore, as shown by FTIR spectroscopy (Figure 2A), theemerging absorption band at 1580 cm�1 can be assigned toN�H bending vibrations of amino groups anchored onMSNs. The successful grafting of HA onto mesoporoussilica was validated by the appearance absorption bands ataround 1650 cm�1 in the sample of MSN-HA, which can beassigned to amide C=O stretching of the carboxyl groupscontained within the attached HA macromolecules. The cor-responding zeta potential was measured in deionized waterat each step. As shown in Figure 2B and Table S1 (Support-ing Information), the zeta potentials of MSP, MSN, andMSN-HA were �13.87, + 19.19, and �38.15 mV, respective-ly. Typically, zeta potentials below �30 mV are consideredas an indication of a colloidally stable system.[16] The strong-ly negative-charged surface enables individual dispersion ofnanoparticles. As shown in Figure S4 and Table S1 (Support-ing Information), MSN-HA exhibited a rather narrow sizedistribution. Moreover, we observed that these nanoconju-gates were stable in phosphate buffered saline (PBS) buffer

Scheme 1. Schematic illustration of the synthesis of HA-capped nanopar-ticles and enzyme responsive release of guest molecules from the host.

Figure 1. TEM micrographs of MSNs (A; scale: 50 nm)) and MSN-HA (B; scale: 100 nm).

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solution (Figure S5, Supporting Information) over a longperiod without any precipitation, which agrees well with thevariation of hydrodynamic diameter of MSN-HA (Figure S6,Supporting Information). Also, no significant variation ofhydrodynamic diameter of MSN-HA was observed in serumcontaining PBS solution (50% fetal bovine serum) duringthe same time (Figure S6, Supporting Information). Thus,the MSN-HA nanoconjugates exhibit long-term stability,which provide additional advantages for blood circulationand the tumor accumulation of nanoparticles by the en-hanced permeation and retention (EPR) effect of well-dis-persed drug carriers.

To investigate the specific enzyme-triggered controlled re-lease of the MSN-HA system, a small amount of dye-loadedMSN-HA (denoted as RhB@MSN-HA) was added to ace-tate buffer (pH 4.5) that contained Hyal-1(150 Uml

�1). Theabsorbance maximum of rhodamine B (553 nm) was plottedas a function of time to generate a release profile. In thecontrol experiment, dye release was also determined byusing suspensions of RhB@MSN-HA under similar condi-tions without Hyal-1. As shown in Figure 3A, the amount ofdye release reached about 82 % within 24 h incubation uponthe introduction Hyal-1. However, only about 9 % releasewas obtained in the same amount of time without Hyal-1,

which thus suggests a good end-capping efficiency and sta-bility under simulated cancer cell conditions. The remarka-ble difference indicated that the induced delivery in thepresence of Hyal-1 was due to the enzymatic-mediated deg-radation and size reduction of the polysaccharide chains atthe surface. To further confirm that enzymes were responsi-ble for the release, two additional experiments were carriedout (Figure 3B). In one experiment, the enzyme was dena-tured by heating enzyme solutions at 90 8C for 45 minbefore addition to the nanoconjugates. As expected, no dyerelease was observed. In another experiment, Hyal-1 wasadded to RhB@MSN-HA after immersion in acetate bufferfor 3 h. Only a small amount of the dye was released withinthe first 3 h, whereas the release of dyes began to increasefollowing the addition of Hyal-1. These results confirmedthat the enzymatic degradation of HA was the mechanismresponsible for the opening of the mesopores. What�s more,only negligible dye release from RhB@MSN-HA at physio-logical pH values (7.4; Figure 3a–c) and 50 % serum con-taining PBS solution (Figure S7, Supporting Information)were monitored, signifying efficient confinement of dye inthe pores of the MSNs by virtue of capping with HA. The

Figure 2. A) FTIR spectra of a) MSP, b) MSN, and c) MSN-HA andB) the corresponding zeta potential measured at each step of the coatingprocess. The error bars represent the standard deviation of three meas-urements.

Figure 3. A) Release profiles of rhodamine B from MSN-HA in the pres-ence of Hyal-1 (a) and without Hyal-1 (b) in acetate buffer (pH 4.5), inPBS buffer (pH 7.4) whitout Hyal-1 (c). B) Responses of the MSN-HAto the deactivated enzyme (a) and delayed release of rhodamine B fromMSN-HA by addition of Hyal-1 solution after incubation for 3 h (b). Theerror bars represent the standard deviation of three measurements.

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J. Ren, X. Qu et al.

potential not only for minimizing drug loss in blood but alsofor enzyme responsive cargo release in tumor tissue by theEPR effect may enhance its overall therapeutic efficacy invivo.

We further extended the controlled release to an in vitrostudy for targeted drug delivery. Targeted delivery to specif-ic cells is entailed in chemotherapy. In our system, the HApolymer was designed to serve as both capping and targetingagent. The cellular targeting efficiency was investigated byincubation NIH3T3 (normal cell) and MDA-MB-231 (CD44over-expressed) cell line with HA conjugated FITC dopedMSNs (FITC-MSN-HA). As shown in Figure 4, althoughthe cellular uptake was observed in both cell lines, MDA-MB-231 cell incubated with FITC-MSN-HA displayed stron-ger fluorescence signals than that of NIH3T3 cells. In thecontrol experiments, weak fluorescence signals were ob-served when both cell lines were incubated with HA-freeMSNs (data not shown). The obvious signal difference sug-gested that HA-attached MSNs were more easily internal-ized by the cancer cells than that of the HA-free nanoparti-cles. This result was attributed to the high specific interac-tion between HA on MSNs and the CD44 receptors overex-pressed on the plasma membrane of the MDA-MB-231cells. All these conclusions demonstrated the efficiency ofour system as drug carriers for targeted therapy.

As to HA catabolism in somatic tissues, HA is tethered tothe cell surface by the combined efforts of CD44 and GIPanchored enzyme Hayl-2, in which HA is cleaved to the20 kDa limit products corresponding to about 50 disaccha-ACHTUNGTRENNUNGride units and then form caveolae. As the caveolae becomeendosomes and finally fuse with lysosomes, the HA frag-ments are degraded into tetrasaccharides by Hyal-1.[11c,14c]

Moreover, different functional-group modified MSNs alwaysshow obvious accumulation in endosomal compartmentsafter endocytosis,[17] which may facilitate the colocalizationof MSN-HA with lysosomes. Also, earlier reports haveshown that HA-based nanomaterials were taken up intocells through CD44-mediated endocytosis and finally accu-mulated in the endosomal and lysosomal compartments,[18]

where HA was degraded by Hyal-1.[14c] Thus, a co-localiza-tion study was carried to investigate the intracellular-uptakeproperties of FITC-MSN-HA. The endosomes and lyso-somes were labeled with red florescent lysotracker upon theincubation of cancer cells with FITC-MSN-HA conjugates(Figure 4B and F). As shown in Figure 4, after 3 h of incuba-tion, the green fluorescence of FITC was readily apparentwithin MDA-MB-231 cells and a significant portion of con-jugates were co-localized with lysotracker red fluorescence,which clearly indicates that the FITC-MSN-HA conjugateshad become highly concentrated within endosomes and lyso-somes of MDA-MB-231 cells. Therefore, after endocytosis,HA is readily degraded into lower molecular fragments byHyal-1 in lysosomal components. Moreover, as shown inFigure S8 (Supporting Information), control experimentstaken at 4 8C showed that weak FITC fluorescence was ob-served. Also, the colocalization of lysotracker and FITC wasnot as obvious as it was at 37 8C. The results indicated that

MSN-HA nanopartilces are taken up inside the cell throughan energy-dependent endocytosis pathway, which is similarto that of HA-liposomes.[18] Taken together, the HA-coatedMSNs showed potential advantage to retard drug releaseoutside cancer cells and facilitated drug release insidecancer cells. In other words, opening of the cap autonomous-ly inside cells of interest could improve anticancer efficiencyand minimize side effects.

Since the RhB-loaded MSNs showed rapid release in thepresence of Hyal-1 which presents at tumor microenviron-

Figure 4. Confocal laser scanning microscopy investigation of the target-ing ability and localization of FITC-labeled MSN-HA to MDA-MB-231cells (targeted cells) and NIH3T3 cells (control cells). Each sample wasimaged for A,E) FITC, B,F) lysotracker, C,G) bright filed, andD,H) merged.

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ment, the high specificity of HA to their receptors shouldprovide efficient and selective cytotoxicity to MDA-MB-231cells. To prove this point, we then investigated the effect oftargeted delivery of chemotherapeutic agent doxorubicin(Dox; Figure 5). Cell viability assays were analyzed by the

MTT assay, which revealed that MSN-HA suspended in PBSwas nontoxic to both cell lines, even at high concentrations,thus indicating that the nanostructures can act as well-de-signed drug carriers. A dose-dependent cytotoxity to bothcells was observed for free Dox and Dox-loaded MSN-HA(Dox-MSN-HA). However, much higher cytotoxity toMDA-MB-231 cells was observed for Dox-MSN-HA thanthat of free Dox at each concentration, which indicated en-hanced killing efficiency. This may be due to the co-effect ofenhanced drug accumulation and the cytotoxity of the nano-carriers. In contrast, relatively weak drug potency of Dox-MSN-HA was observed for NIH 3T3 cells, which wasmainly attributed to two reasons 1) lack of CD44 receptors

on the cell surface and 2) a lower level of intracellular Hyal-1.[12e, l] Although endogenous HA correlating with and ac-tively promoting tumor progression may compete for the re-ceptor to some extent, HA turnover occurs more rapidly inmalignant tissues owing to the high levels of Hyals.[11a]

Moreover, work involving HA-conjugated nanomaterialshas shown excellent tumor-targeting ability for drug/genedelivery or imaging in vivo.[12d,e, g, l] Taken together, we con-firm our hypothesis that HA-capped MSNs could serve asnanoreservoirs in an enzyme-responsive controlled-releasepathway for targeted drug delivery.

Conclusion

In summary, we have demonstrated, as a proof-of the-con-cept and for the first time, that the use of HA as both cap-ping and targeting agent on the surface of MSNs provides asuitable method for the design of an enzyme-responsivenanoreservior for efficient targeted drug delivery to cancercells. Cargo release was triggered by specific enzymes pres-ent in the tumor microenvironment. In vitro studies haveshown the feasibility of using this nanocarrier as a targetedcontrolled drug delivery system in cancer cells with remark-ably enhanced killing efficiency. More importantly, the ex-cellent biocompatibility, cancer cell recognition ability, andefficient intracellular drug release provide a basis for in vivocontrolled-release in biomedical applications and cancertherapy.

Experimental Section

Materials and instrumentation: Nanopure water (18.2 MW; Millpore Co.,USA) was used in all experiments and to prepare all buffers. Tetraethy-lorthosilicate (TEOS) was purchased from Sigma–Aldrich. 3-(2-Amino-ACHTUNGTRENNUNGethylamino)propyltrimethoxysilane was obtained from Alfa Aesar.Sodium hyaluronate (MW�100 KDa) was purchased from Aladdin. Allthe chemicals were used as received without further purification. Hyal-ACHTUNGTRENNUNGuron ACHTUNGTRENNUNGidase from bovine vitreous humor was purchased from ShanghaiSangon Biological Engineering Technology & Services (Shanghai,China).

General techniques : FTIR analyses were carried out on a Bruker Vertex70 FTIR Spectrometer. SEM images were obtained with a Hitachi S-4800FE-SEM. N2 adsorption–desorption isotherms were recorded on a Micro-meritics ASAP 2020 m automated sorption analyzer. TEM images wereacquired with a TECNAI G2 transmission electron microscope (PhilipsElectronic Instruments Co. the Netherlands) at 200 kV.

Cargo loading and HA capping experiments : For rhodamine B loading,the MSNs (60 mg) were soaked in a solution of rhodamine B (10 mg) for24 h. The nanoparticles were then centrifuged and washed thoroughlywith pure water to remove unloaded and adsorbed molecules. HA(30 mg) was hydrated in water overnight and then activated by usingEDC (2 mg mL�1) and NHS (2 mg mL�1) in a 2-(N-morpholino)ethane-sulfonic acid (MES) buffer (pH 6.0) for 30 min. Twenty microliters ofPBS buffer (100 mm, pH 7.4) was then added to the mixture, followed bythe addition of 12 mg dye loaded MSNs at room temperature with con-tinuous stirring for 20 h. The unreacted HA was removed by centrifuga-tion. All the washing solutions were collected, and the loading of rhoda-mine B was calculated from the difference in concentrations of the initialand dyes left to be approximately 53.3 mmol g�1 SiO2. For Dox loading

Figure 5. In vitro viability of MDA-MB-231 cells (targeted cells) (A) andNIH3T3 cells (control cells) (B) in the presence of free Dox, Dox-MSN-HA and corresponding MSN-HA nanoconjugates. The error bars repre-sent the standard deviation of three measurements. &: MSN-HA; *: freeDox; ~: Dox-MSN-HA.

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J. Ren, X. Qu et al.

and capping process, the MSNs (20 mg) were soaked in a solution of Dox(4 mL, 1 g L�1) in nanopure water for 24 h. The loading of Dox was calcu-lated be approximately 68.5 mmol g�1 SiO2.

Dye release experiments : Rhodamine B loaded MSN-HA (10 mg) mate-rial was dispersed in aqueous buffer solutions (5 mL, pH 4.5 acetatebuffer) with and without Hyal-1 (150 Uml

�1) at 37 8C. Aliquots (50 mL)were taken from the suspension at predetermined time intervals, and re-placed with an equal volume of the fresh medium. The release of rhoda-mine dye from the pore to the buffer solution was monitored by the ab-sorbance band of the dye centered at 553 nm.

Confocal laser scanning microscopy (CLSM): FITC exhibits intensegreen fluorescence under UV light. This property allows the use of fluo-rescence to study the distribution of FITC-MSN-HA inside the cells.MDA-MB-231 and NIH3T3 cells, incubated with nanoparticles inmedium for 2.5 h, were next washed three times with PBS and transfer-red to serum-free medium and examined by CLSM. For the co-localiza-tion study, 10 mm lysotracker was added for specific staining of the lyso-somes of cancer cells during the last 0.5 h of the 3 h incubation afterwashing free nanoparticles. The remaining experimental procedures werethe same as those for the internalization study.

Cytotoxicity assays : MTT assays were used to probe cellular viability.MDA-MB-231 and NIH3T3 cells were seeded at a density of 5000 cells/well (90 mL total volume/well) in 96-well assay plates. After 24 h, drugs atthe indicated concentrations were added and cells were further incubatedfor 48 h. To determine toxicity, MTT solution (10 ml, BBI) was added toeach well of the microtiter plate and the plate was incubated in the CO2

incubator for an additional 4 h. The cells then were lysed by the additionof DMSO (100 ml). Absorbance values of formazan were determinedwith Bio-Rad model-680 microplate reader at 490 nm (corrected forbackground absorbance at 630 nm). Six replicates were done for eachtreatment group.

Acknowledgements

Financial support was provided by the National Basic Research Programof China (2011CB936004 and 2012CB720602) and the National NaturalScience Foundation of China (Grants 20831003, 90813001, 20833006,90913007, 21072182).

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Received: June 8, 2012Revised: November 8, 2012

Published online: January 9, 2013

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