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The Effects of Spacer Length and Composition on Aptamer‐Mediated DOI:10.1002/cbic.201600092 Communications The Effects of Spacer Length and Composition on Aptamer-MediatedCell-Specific Targeting with Nanoscale PEGylated Liposomal Doxorubicin Hang Xing+,[a] Ji Li+,[a] Weidong Xu,[c] KevinHwang,[a] Peiwen Wu,[a] Qian Yin,[b] Zhensheng Li,[a] JianjunCheng,[b] and Yi Lu*[a] Aptamer-based targeted drug delivery systems have shown tissues.[1] Among manyphysical properties, the surfaceproper- significant promise for clinical applications. Althoughmuch ties of nanocarriers play acentralrole, as the interface be- progress has been made in this area, it remains unclear how tween the delivery vehicle and the biological componentsin PEG coating would affect the selective binding of DNA aptam- and on cells during the delivery process determines the stabili- ers and thusinfluence the overall targeting efficiency.To ty,targeting ability,and kinetics of drug release of the nanocar- answer this question, we herein report asystematic investiga- riers.[1f, 2] Therefore, nanocarrier surfaces have been functional- tion of the interactions between PEG and DNA aptamerson ized with active biorecognition agents that can bind to specific the surface of liposomes by using aseries of nanoscale liposo- receptors on the cell surface, allowing selective accumulation mal doxorubicin formulationswith different DNA aptamer and of nanomedicine into diseased tissues butnot normaltissues.[3] PEG modifications. We investigated how the spatial size and Among the types of biorecognition agentsutilized to en- composition of the spacer molecules affected the targeting hance selectivity of nanocarriers, DNA aptamers, which are ability of the liposomedelivery system.Weshowedthat single-stranded oligonucleotides that can selectively bind to aspacerofappropriate length was criticaltoovercome the targetmolecules, have emerged as apromising class of target- shieldingfrom surrounding PEG molecules in order to achieve ing ligand due to their higher stability and lower cost than an- the best targeting effect, regardless of the spacer composition. tibodies, another common class of biorecognition agent for Our findings provideimportant guidelines for the design of targeted therapy. Moreover,aptamers against different targets aptamer-based targeted drug delivery systems. of interest can be selected through the systematic evolution of ligandsbyexponential amplification (SELEX) process, providing the possibility of developing nanomedicines for specific cells Pharmaceutical drug delivery systems on the nanometer scale and tissues.[4] These features, together with non-immunogenic- have been widely used as nanocarrierstoboost drug efficacy. ity and ease of preparation and functionalization onto nano- When compared with their free drugcounterparts, drugs en- carriers,make aptamers an excellent choice as aclass of biore- capsulated in nanocarrierscan display higher stability, fewer cognition agents fornanomedicine.[5] Indeed, many DNA apta- side effects, and more controllable release kinetics. In order to mer-functionalized nanocarriers have been used forsuccessful maximize their therapeutic potential, atremendous amount of delivery of drugs both in vitro and in vivo.[5a,6] effort has been devoted toward studying how the physical Despite the enhanced selectivity stemming from specific sur- properties of nanocarriers, such as size, shape, rigidity,charge, face interactions between biorecognition molecules, such as and surface function, can influence their interactions with tar- aptamers, and the target receptors on the disease cells, the geted cells and thus the efficiency of deliverytothe relevant targeted delivery process is complicated by other biological components under physiological conditions.[7] Anumber of lit- + + [a] H. Xing, J. Li, K. Hwang, P. Wu, Z.Li, Prof.Y.Lu erature reports have demonstrated that serum proteins can Department of Chemistry,Beckman Institute for AdvancedScience and [8] Technology absorb onto nanoparticle surfaces to formaprotein corona. University of Illinois at Urbana–Champaign Nonspecifically adsorbed serum proteins affect the physiologi- 405 NMathewsAvenue, Urbana, IL 61801 (USA) cal properties of nanocarriersand can mask their targeting ca- E-mail:[email protected] pabilities toward cell receptors, thereby leading to aloss of [b] Q. Yin, Prof. J. Cheng recognition specificity and fast clearance by the reticuloendo- MaterialScience and Engineering, Beckman Institutefor Advanced Science [9] and Technology thelialsystem (RES). To suppress nonspecific serum protein University of Illinois at Urbana–Champaign adsorption,antifouling polymers such as polyethylene glycol 405 NMathewsAvenue, Urbana, IL 61801 (USA) (PEG) have been used to backfill the surface of nanocarriers, al- [c] W. Xu lowing amuch longercirculation time in blood and ahigher Department of Chemistry,Tsinghua University chance of nanocarrier accumulation in the desired tissue.[10] Al- Beijing 100084 (China) thoughthe use of PEG modificationstoinhibit protein adsorp- [+] These authors contributed equally to this work. tion has been widely reported, the interaction between back- Supporting information and the ORCID identification number(s) for the au- thor(s) of this article can be found under http://dx.doi.org/10.1002/ filling PEG molecules and neighboring recognition molecules cbic.201600092. on the same surface has not been thoroughly studied.[5a,6c] A This manuscript is part of aSpecial Issue on DNA Nanotechnology. few studies have reported that the PEGshield on the nanopar- ChemBioChem 2016, 17,1111–1117 1111 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications ticle surfacecan sterically hinderthe binding of antibodies or peptides to areceptor target, and appropriate backfilling PEG length is critical to achieve optimal cellular uptake.[2a,11] In order to design aptamer-based nanoscale drug delivery systemsfor effective clinicalapplications,itisimportant to carry out systematic studies of the interactions betweenthe targetingaptamersand the backfilling PEG molecules in order to find the optimal parameters to maximizethe targeting effect while minimizing non-specific interactions. To achieve this goal, we used aPEGylated liposomaldoxorubicin (PEG-Lip- Dox) with aDNA aptamer attached to the surface of the lipo- some as the modelsystem to investigate the effect of spacer length and PEG modification on aptamer-mediated cell-specific targeting. We choose liposomes for this investigation, because they are one of the most successful drug delivery systems due to inherentadvantages such as high drug-loading efficiency and biocompatibility.[5a,6c,12] Furthermore, aPEG-Lip-Dox, Doxil, was the first nanomedicine formulation approved by the United States Food and Drug Administration (FDA) for treating avariety of cancers.[13,14] Therefore, the knowledge gained from this study can be readily translated into clinical applica- tions. In previous studies, we have demonstrated that aPEGylated liposomesystem containing anticancer drugs such as cisplatin or Dox can be effectively functionalized on its surface with aDNA aptamer called AS1411,whichcan selectively recognize nucleolin (NCL), an mRNA-stabilizing protein overexpressed on the plasma membrane of many types of cancer cells, including Figure 1. Schematic of the targeting abilityofPEGylated liposomaldoxoru- breast cancer cells.[5a,6c] We furthershowedthat this targeted bicin (PEG-Lip-Dox), modified with DNA aptamerAS1411byusing oligo-T se- liposomal Dox construct was able to facilitate selective drug quences of different lengths as spacers, towards breastcancer cells through specific binding of AS1411tonucleolin on cell membranes. HSPC, cholester- uptake into breast cancercells[5a] and allowed enhanced tumor ol, and mPEG-DSPE (mPEG2000-DSPE or mPEG5000-DSPE) were combined in penetration and antitumor efficacy in MCF-7 tumormodelsin a2:1:0.16 molar ratio. Cholesterol-modified DNA strands were immobilized mice.[6c] Although these results are encouraging, it remains un- onto the liposomal surfacebyintercalating the 3’ cholesterol modification clear how the PEG coating would affect the selective binding into the lipid bilayer. of the NCL target by the DNA aptamer and thus influence the overall targeting efficiency. To clarify this issue, we report herein asystematic investigation of the interactions between total lipid content (Figure 1). The combination of HSPC and PEG and DNA aptamersbypreparing aseries of liposomal Dox cholesterol provides liposomes with ahigh phase transition formulations with differentDNA aptamer and PEG modifica- temperature of 488C, as well as high rigidity and low permea- tions, as well as adetermination of how the spatial size and bility under physiological conditions.[15] Dox, an anticancer composition of the spacer molecules on the surface of the lip- drug as well as afluorescent probe,was encapsulated into the osome affect the targeting ability of the liposomedelivery liposomethrough apH–ion gradientloading method.[16] This system toward breastcancer cells. well-establishedformulation of PEGylated liposomes has been The formulation of the liposomal Dox with PEG and aptamer used in clinical practicefor cancertreatment for decades. To in- modifications was based on Doxil, an FDA-approved, Dox-con- troducethe targeting ability of PEG-Lip-Dox, AS1411, a26-mer taining liposomewith no targeting ligand incorporated.[14] We NCL aptamer(Apt) with the sequence 5’-GGT GGT GGT GGT modifiedthis liposomewith different aptamers to make it TGT GGT GGT GGT GG-3’ was used as atargeting agent
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