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Cite this:Chem. Commun., 2014,

50, 1579

Multifunctional nanoparticles via host–guestinteractions: a universal platform for targetedimaging and light-regulated gene delivery†

Wenyu Li, Jianwei Du, Kun Zheng, Peng Zhang, Qiaoling Hu and Youxiang Wang*

Host–guest assembly provides a universal platform to construct responsive

carrier systems for targeted imaging and controllable gene delivery. The

best advantage of this strategy is that systems are very easy to handle, do

not involve tedious chemical reactions and can be flexibly optimized by

changing the functional tags responding to a request.

Nanobiotechnology has been advancing rapidly toward biomedicalapplications of nanoparticles in cancer diagnosis and therapy.1 Overthe past decade, multifunctional groups have been covalently con-jugated to smart carrier systems combining diagnostic detectionagents and therapeutic payloads (anticancer drugs, siRNA, etc.),which are exciting for their potential applications in the earlydetection and treatment of human cancer.2 Many researchers haveaspired to develop responsive carrier systems for the controllablerelease of therapeutic agents that are sensitive to environmentalstimuli such as temperature, pH, redox or specific biomolecules.3

However, the covalent synthetic steps are usually complicated andtime-consuming. In some cases, multiplex synthetic progress mightbe completely carried out again on changing one aspect such as animaging agent or ligand. Recently, supramolecular chemistry hassparked the emergence of the modular assembly of different func-tional tags such as imaging, targeting, and treating modules. Typicalsupramolecular host b-cyclodextrin (b-CD) has well-known host–guest interactions with a vast array of hydrophobic compounds,such as azobenzene (Az), adamantane (AD), and cholesterol.4 Thelight-controlled molecular recognition of CD for Az has beenexploited to develop light-responsive drug or gene delivery.5 Forexample, a ternary supramolecular system was developed based on alight-responsive Az–CD host–guest interaction, which can reversiblycapture and release DNA.4b Meanwhile, our recent study also foundintracellular dePEGylation could be easily controlled via light-regulated host–guest interactions between b-CD and Az.5c Therefore,host–guest assembly can provide a novel strategy to physically

integrate diagnostic imaging agents and responsive gene deliverycarriers.

Herein, we attempted to construct multifunctional nanoparticlesvia host–guest interactions between b-CD and its guest molecules(Fig. 1). Branched polyethylenimine (PEI) was selected as a scaffoldpolymer and b-CDs were conjugated as host units. Galactose (Gal), atargeting moiety of asialoglycoprotein receptor (ASGP-R) overexpressedon the surface of hepatocytes, was conjugated on azobenzene-polyethylene glycol (Az-PEG-Gal). Fluorescein isothiocyanate (FITC)was selected as a fluorescence imaging probe and conjugated onadamantane (AD-FITC). Subsequently, the supramolecular polymerwith multi-functions was easily constructed by host–guest assemblyand then used to load therapeutic agents (e.g., DNA) through theelectrostatic interactions. The PEG shell was expected to equipthe multifunctional nanoparticles with excellent stability. Specificrecognition promoted the multifunctional nanoparticles to accumu-late at the disease site, which facilitated non-invasive bioimaging.1b

The light-responsive CD–Az host–guest interaction might facilitate thecontrollable release of DNA inside the cells. Therefore, the host–guestassembly acted as a flexible, universal platform to construct multi-functional nanoparticles that physically incorporated targeted imagingand controllable gene delivery into one package.

Firstly, PEI-CD was synthesized by the substitution reaction of6-deoxy-(p-toluenesulfonyl)-b-CD (6-OTs-b-CD) with the amine groupsof PEI.6 The CD-grafting level confirmed by 1H NMR was 1.5%,indicating that every PEI chain had 9 CD molecules (Fig. S1, seeESI†). As shown in Scheme S1 (see ESI†), Az-PEG-Gal and AD-FITCwere synthesized. In addition, it has been documented that theassociation constant Ka of b-CD to AD or Az was 2–4 � 104, 1.7 �103 M�1, respectively.7 Due to the significant difference in Ka values,AD-FITC was firstly incorporated onto PEI-CD with the molar ratio ofAD to CD at 1 : 3, followed by the addition of Az-PEG-Gal to avoidcompetitive inclusion among different guests. Next, Az-PEG-Gal wasincorporated with various molar ratios of Az on Az-PEG-Gal to b-CDsremaining on PEI-CD (Az/RC). The protocol of inclusion in detail wasmonitored according to our previous study.5c Then, the resultingsupramolecular polymers AD-FITC/PEI-CD, AD-FITC/PEI-CD/Az-PEGand AD-FITC/PEI-CD/Az-PEG-Gal were successfully obtained via

MOE Key Laboratory of Macromolecular Synthesis and Functionalization,

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou

310027, P. R. China. E-mail: yx_wang@zju.edu.cn; Fax: +86 571 87953729;

Tel: +86 571 87953729

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc48098d

Received 22nd October 2013,Accepted 20th November 2013

DOI: 10.1039/c3cc48098d

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1580 | Chem. Commun., 2014, 50, 1579--1581 This journal is©The Royal Society of Chemistry 2014

host–guest interactions in mild conditions and designated as SP,SPG and SPG-Gal, respectively.

Driven by the electrostatic interactions, the multifunctionalsupramolecular polymers condensed DNA into nanoparticles. Theparticle sizes and z-potentials were evaluated in detail by ZetasizerNanoseries. As shown in Fig. S2 (see ESI†), it was found that the sizesand zeta-potentials decreased slightly with Az/RC ratio increment,and finally had a very small z-potential value of about 6 mV. Theresults suggest that the nanoparticles formed core–shell architec-tures with a hydrophilic and neutral PEG-Gal shell surrounding theparticle core, which provided feasibility of targeted cellular uptake.8

The Az/RC ratio was kept at 4 in the next experiments to maintainsufficient PEG shell for colloidal stability. Fig. 2(a) shows thenanoparticles have a tightly packed structure. The fluorescenceimage shown in Fig. 2(b) indicates that the particles were observedwith bright green fluorescence, suggesting that the multifunctionalnanoparticles could be used in intracellular trafficking. Moreover, anagarose gel retardation assay indicated that the incorporation ofFITC and PEG-Gal via host–guest interactions did not significantly

affect the DNA condensation ability (Fig. S3, see ESI†), which is ofbenefit for transfection.

The long blood circulation of nanoparticles is a prerequisite forcancer diagnosis and gene therapy in vivo.9 Dynamic light scattering(DLS) and TEM revealed that the multifunctional nanoparticles werestable in physiological salt conditions and complete cellular culturemedia containing 10% fetal bovine serum (Fig. S4, see ESI†), whichwas ascribed to steric repulsion of sufficient PEG shell. Heparin wasused as a counter polyanion to evaluate the in vivo stability againstanionic proteins in the bloodstream. As shown in Fig. 2(c) and (d),for PEI/DNA nanoparticles, the free DNA band began to emerge at aheparin concentration of 60 mg mL�1. In contrast, in case of SPG-Gal/DNA nanoparticles, no free DNA bands were observed even at theheparin concentration of 80 mg mL�1. The improved competitionstability can be attributed to the steric repulsion of the PEG shell,preventing the access of counter polyanion. On the other hand, thelight-regulated host–guest interaction Az–CD was expected to equipthe multifunctional nanoparticles with PEG-detachable ability forfacilitated DNA release. Next, the z-potential was investigated afterlight irradiation at 365 nm for 15 min. It was found that lightirradiation significantly increased the z-potential and particle size(Fig. S2, see ESI†), which were comparable to those of SP/DNAnanoparticles. Moreover, after irradiation, the fluorescent intensity ofDNA bands was improved in the agarose gel chamber (Fig. S5, seeESI†), which indicated that the 15 min light irradiation was effectivefor PEG detachment from the multifunctional nanoparticles.

The ideal multifunctional nanoparticles should actively target andaccumulate at the cancer site, followed by non-invasive bioimagingdiagnosis and controllable gene therapy. In this research, HepG2(human hepatoblastoma cell line) with the ASGP-R overexpressed onthe cell surface was used to investigate the specific-targeting cap-ability. The multifunctional nanoparticles incorporated with FITC viahost–guest interactions were directly used for cellular uptake, whichcould be detected by flow cytometry. As shown in Fig. 3(d), the PEGshell hindered the cellular uptake of SPG/DNA nanoparticles com-pared with that of SP/DNA nanoparticles. However, the cellularuptake profiles of SPG-Gal/DNA nanoparticles were approximately4 times for 0.5 h and 1.2 times for 4.5 h higher than those ofnanoparticles without targeting molecules. The results suggest thatthe multifunctional nanoparticles were efficiently accumulated andtaken up by cancer cells based on specific recognition.

As a direct proof of concept, we carried out the bioimaging andintracellular trafficking of the multifunctional nanoparticles byconfocal laser scanning microscopy (CLSM). Cy3-DNA was used tobetter understand intracellular trafficking. As shown in Fig. 3(a) and(b), for SP/DNA nanoparticles, part of Cy3-DNA was observed to enterinto the nuclei, although there was some part of overlapped yellowfluorescence inside the cells. In the case of SPG-Gal/DNA nano-particles, effective endocytosis was achieved via specific recognition,with obvious yellow fluorescence, and mainly distributed in thecytoplasm. Combined with the results of competition stability inheparin, we speculated that the PEG shell hindered the disassemblyof nanoparticles inside the cells. The stable CD–AD host–guestinteraction was expected to maintain FITC to monitor the distribu-tion of the polycations. Fig. 3(c) indicates that after 15 min lightirradiation (l = 365 nm), most of DNA with red fluorescence was

Fig. 1 Illustration of the multifunctional nanoparticles developed via host–guest interaction for targeted imaging and controllable DNA delivery.

Fig. 2 TEM (a) and fluorescence (b) images of SPG-Gal/DNA nanoparticles(N/P = 10). The observation was performed using a 40� objective; agarosegel electrophoresis of PEI/pDNA (c) and SPG-Gal/pDNA (d) nanoparticles(N/P = 10) in the presence of various concentrations of heparin.

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localized in the nuclei and polycations with green fluorescence weremainly distributed in the cytoplasm. The results provide direct proofthat the light-regulated PEG detachment facilitated DNA release toenter into nuclei. Here, if only FITC was replaced with NIR opticalimaging agents such as Cy5.5, the nanoparticles would be easilyvisualized by NIR optical imaging in vivo to realize a diseasediagnostic application.

Next, the cytotoxicity of the multifunctional nanoparticles andlight irradiation were investigated. As shown in Fig. S6 (see ESI†), thecytotoxicity of the multifunctional nanoparticles was much lowerthan that of PEI/DNA particles mainly due to the well-knownbiocompatibility of cyclodextrin and PEG.10 More importantly, light(l = 365 nm) irradiation for 15 min had little effect on cell viability.Encouraged by these results, luciferase plasmid DNA pGL-3 was usedas a model reporter gene to investigate the therapeutic ability. Asshown in Fig. 3(e), SPG/DNA nanoparticles showed the lowesttransfection efficiency, which was attributed to the reduced cellularuptake and hindered ability of intracellular DNA release. However,when incorporated with targeting molecules, SPG-Gal/DNA nano-particles showed around 2 orders of magnitude higher transfection

efficiency than the nanoparticles without targeting molecules. Themain reason was that specific recognition increased cellular uptakeefficiency. Furthermore, light-stimulus equipped the multifunctionalnanoparticles with around 1.5 times higher transfection efficiencythan that without light irradiation, which was comparable to SP/DNAnanoparticles. From these results, we confirmed the feasibility ofSPG-Gal/DNA nanoparticles as a responsive multifunctional system.

In summary, novel multifunctional nanoparticles were success-fully constructed via host–guest interactions. The best advantage ofthis strategy is that the system is very easy to handle, does not involvetedious chemical reactions and can be flexibly optimized bychanging the functional tags responding to a request. We believethe host–guest assembly provides a universal platform to constructresponsive carrier systems for targeted imaging and controllablegene delivery, which might have potential application in futuredisease diagnosis and therapy.

This work was financially supported by the National NaturalScience Foundation of China (21074110, 51273177).

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Fig. 3 CLSM images of HepG2 cells exposed to (a) SP/Cy3-DNA, (b) SPG-Gal/Cy3-DNA, and (c) 15 min light irradiation treatment with SPG-Gal/Cy3-DNA after 4.5 h incubation, followed by another 12 h incubation in theabsence of nanoparticles. The nuclei were stained with DAPI (blue).(d) Cellular uptake profile of HepG2 cells exposed to different nano-particles for 0.5 h and 4.5 h. (e) In vitro transfection of the luciferase geneinto HepG2 cells mediated by different nanoparticles for 48 h. *denotesstatistically significant difference at p o 0.05.

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