Inducible Epitope Imprinting: ‘Generating' The Required Binding Site In Membrane Receptor For Tumor Targeted Drug Delivery

Nowadays,in targeted drug delivery,certain cell-surface receptor overexpressed in tumor are selected as targets and the corresponding ligands(e.g.,antibodies,peptides,aptamers)are used as targeting moieties and conjugated onto the surface of drug carriers,and this is one of the main current strategies used for designing targeted drug delivery systems.However,membrane receptors constitute a challenging class of targets for ligand screening because of limitations in the production of sufficient amounts of soluble,functional,and stable receptors.Alternatively,the molecularly imprinted polymeric nanoparticles(MIPNPs)represent a potential promising type of ‘artificial ligands' toward membrane receptors,which can be prepared by using an epitope to replace the whole target protein as template,therefore is cost-and time-saving.Similar to antibodies,MIPNPs can bind to a conformational epitope with much higher affinity and specificity than their linear counterpart because of the epitope's specific three-dimensional shape,offering a great possibility that designed MIPNPs could selectively recognize a specific structured region of target cell receptor and subsequent cells as a novel TDDS.Previously,we reported active drug targeting to tumors through“conformational epitope imprinting”: an entire target receptor,p32,was replaced with a structured peptide epitope as template in the molecular-imprinting process,and a polymeric “magic bullet” was obtained.This strategy could facilitate the recognition of other proteins,given the premise that binding domains are conformationally rigid.However,the N terminal of p32 is ?-helix in conformation,for flexible “non-epitopes,”this structure-based molecular imprinting is inadequate and unsuitable.As a membrane-bound protein,folate receptor-?(FR?)is overexpressed in several human cancers,and the N terminal of FR? is flexible.Based on this,our study puts forward an inducible epitope imprinting strategy.The molecular imprinting nanoparticles(MIPNP-F)was synthesized on the basis of ?-helix FN peptide(the N terminal of FR?)as the template.The imprinted nanoparticles with formative “hole” can specifically recognize,innduce the N terminal of FR? on the cell membrane and band it,then can effectively target the tumor.More interestingly,the N-terminal disordered region of FR?was distant from the folate-binding pocket,therefore,the free folic acid(FA)conducts no competitive inhibition for the target ability of MIPNP-F.In the first chapter,the feasibility analysis of inducible epitope imprinting strategy was conducted.The imprinted nanoparticles with ?-helix “hole”(MIPNP-A)and linear“hole”(MIPNP-L)was synthesized by using the apamin(an ?-helical peptide stabilized by 2 disulfide bonds))and linear apamin(the linear derivative of apamin,4 Cys replaced with Ala)as the template,respectively.When incubated with MIPNP-A,the original disordered structure of linear apamin was clearly changed to ?-helix,measured by circular dichroism(CD)spectra,Intriguingly,MIPNP-A also recognized linear apamin,with a binding trend similar to that of apamin.By contrast,MIPNP-L showed very low response to apamin.These results demonstrated for the first time that polymeric NPs generated by imprinting a structured peptide can potentially induce the expected conformational change in the linear derivative with a corresponding binding ability;To futher study the inducible epitope imprinting strategy used for tumor targeted drug delivery.Here,the N-terminal of FR was selected as the target.FN peptide was synthesized,and it can be forced into ?-helix in 50% TFE/Water.The molecular-imprinting reaction was conducted in 50% TFE/water solution,the FN peptide folded into ?-helix as the structured template,and acrylamide,2-(trifluoromethyl)acrylic acid served as the functional monomers,N,N?-methylene bisacrylamide and trifluoro ethyl acrylate used as crosslinking agent.The obtained nanoparticles with ?-helix “hole”(MIPNP-F)exhibited a mean particle size of(78.3±2.5)nm,with the polydispersity index(0.175±0.023).The morphology of nanoparticles observed by scanning electron microscope(SEM)and transmission electron microscopy(TEM)revealed that MIPNP-F is spherical in form with uniform distribution.The above reaults demonstrated that the MIPNP-F was prepared successfully.This provides a favorable starting point for inducible epitope imprinting strategy used for tumor targeted drug delivery.In the second chapter,the targeting ability and carrier safety in vitro and in vivo was investigated.The ?-helix content of FN peptide was 36% upon MIPNP-F binding,compared with 5% for the peptide alone.And as expected,MIPNP-F can more tightly bound to linear FN peptide compared to NIPNP.NP-cell interaction was monitored in real time and the KD values(2.81E-8±5.81E-10)were obtained on HeLa cells treated with MIPNP-F compared to(2.15E-5±3.05E-7)obtained on the cells treated withNIPNP.On the HeLa,KB,and PANC-1 cell line(FR?-positive cell),the uptake of MIPNP-F was significantly higher than that of NIPNP,as observed by fluorescence microscope and quantified using flow cytometry.The fluorescence intensity of MIPNP-F groups was 3.89,4.05 and 3.46 times higher than NIPNP groups.On the FR low expression cell line A549 and MCF7,there was no difference on the cell fluorescence intensity of MIPNP-F groups and NIPNP groups.MIPNP-F mainly uptaked into cells through the caveolar pathway,which agrees with the cell uptake mechanism of FA modified nanoparticles(FA-NP)and the reported FR-mediated endocytosis.We also verified the targeting ability of MIPNP-F in vivo.When intravenously injected into the tumor bearing nude mice,MIPNP-F accumulated in tumors with a much higher level than the NIPNP treated group.We also prepared NPs encapsulating a photosensitizer,methylene blue,MIPNP-F showed drastically stronger inhibitory effect on HeLa cells and HeLa subcutaneous tumors than NIPNP in vitro and in vivo.What's more interesting,in vitro,high concentration of FA showed no effect on the Hela cell uptake of MIPNP-F.And in vivo,in the case of a pre-injection of free FA near the tumor,the specific targeting ability of MIPNP-F was also maintained.By constract,the targeting ability of FA-NP was sensitive to free FA both in vitro and in vivo.Moreover,the cytotoxicity assays and pathology analysis of main organs in mice primarily demonstrated the high bio-compatibility of MIPNP-F.All the above results suggesting that MIPNP-F holds great potential as a tumor targeted drug delivery agent.In conclusion,based on the conformational epitope imprinting,we explored the inducible epitope imprinting strategy,and suggesting that the unstructured N-terminal region of FR? can be reconstructed as a real epitope by the customized nanoparticles,and thus used for an active tumor targeted drug delivery agant both in vitro and in vivo.The inducible epitope imprinting strategy work out the difficulties of untargetable certain flexible protein domains,enriched the content of receptor recognition,broaden the application of the molecular imprinting technique and current understanding of smart vehicles in tumor targeted drug delivery.