A Biohybrid Lurker-to-Attacker Strategy To Solve Inherent Dilemma of

Feb 15, 2017 - A Biohybrid Lurker-to-Attacker Strategy To Solve Inherent Dilemma ... forward a biohybrid strategy to solve this long-standing dilemma ...
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A Biohybrid Lurker-to-Attacker Strategy To Solve Inherent Dilemma of Positively Charged Delivery Nanoparticles Jingyi Zhu, Mingkang Zhang, Diwei Zheng, Bin Yang, Ning Ma, Runqing Li, Jun Feng,* and Xianzheng Zhang Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China S Supporting Information *

ABSTRACT: Nonspecific cell attack and rapid in vivo recognition/ clearance have been the unsurmountable hurdles against the application of positively charged nanoparticles (pcNPs). The frequently used active targeting approach by grafting specific ligands onto pcNPs suffers from their strong electrostatic interaction with normal cells. We herein put forward a biohybrid strategy to solve this long-standing dilemma in the development of tumor-specific pcNPs. pcNPs are arranged to put on a biological “coat” derived from cancer cell membranes. This design renders pcNPs the high recognition to the homotypic cancer cells with even higher uptake efficiency than the parent pcNPs, while considerably inhibiting the adsorption by biological components, the macrophage capture, and the uptake by the heterotypic cells (e.g., normal and macrophage cells). Encouragingly, the tumor self-targeting by coating pcNPs with the cancer cell membranes proved to be achievable, allowing the role transition to an “attacker” upon reaching the homologous tumor developed from the source cancer cells. This approach paves a facile way to overcome the current limitations for in vivo application of pcNPs.

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double-edged sword ascribed to the inherent characteristic of pcNPs has been a hardly surmountable hurdle against the translation of pcNPs into practical applications. Recently, an approach termed “tumor environment responsive charge conversion” has been put forward in attempts to address this challenge.9,10 The initially negatively charged NPs act as a “lurker” in normal conditions while undergoing transformation to an “attacker” at tumor sites owing to the locally bioresponsive conversion to pcNPs.11,12 In a like manner, two mechanisms termed “pop-up”13 and “presentation”14 have been raised to make pcNPs selectively attack tumor cells. The positively charged moieties are initially buried in the inner domain of NPs while being exposed onto the NP periphery to recover the excellent cell-uptake ability in a tumor-environment-responsive fashion. In these delicate designs, the extreme sensitivity to tumor matrix must be needed; however, it is generally unsatisfactory. For instance, tumorous extracellular pH has been intensively used to trigger the recovery of positive charge of NPs15 because of the Warburg effect-induced acid microenvironments (∼pH 6.2−6.9) in various tumors.16 However, the subtle difference from the normal microenvironment (pH 7.2−7.4) makes it very difficult to fulfill the pHresponsive conversion as expected.17 On the other hand, the

anoparticle (NP)-mediated delivery of various therapeutics and imaging agents shows strong potency to drastically enhance the efficacy for many illnesses, particularly for tumor diseases. Among those, positively charged NPs (pcNPs) display unparalleled advantages owing to their inherently excellent capabilities of carrying negatively charged agents (e.g., drug, siRNA, DNA) and penetrating targeted cells.1 As one notable example, nonviral cationic gene-embeded pcNPs afford high transfection performance in most of the cell lines.2 Besides, micellar pcNPs exhibit extraordinarily strong loading capacity for the anion-containing drugs (i.e., carboxyl and sulfonate, etc.).3 Hence, great efforts have been made to develop clinically applicable organic and inorganic pcNPs.4 Unfortunately, the majority of pcNPs work well in vitro but fall short in vivo. The major cause also arises from pcNP’s positive charge that would induce easy adsorption by the oppositely charged blood components, nonspecific uptake by normal cells, rapid recognition and accelerated clearance by the defense mechanism, and even the risk of thrombosis/embolism.5 Consequently, there occurs the poor availability of therapeutic payloads at the diseased sites along with the deep penetration in most organs, as detected after systemic injection of pcNPs.6 Although surface modification with certain ligands is well established to endow NPs with the active targeting function,7 the specific ligand−receptor recognition seems to difficultly surpass the strong nonspecific interaction between pcNPs and cells, resulting in insignificant improvements for pcNPs.8 This © 2017 American Chemical Society

Received: December 2, 2016 Revised: February 14, 2017 Published: February 15, 2017 2227

DOI: 10.1021/acs.chemmater.6b05120 Chem. Mater. 2017, 29, 2227−2231

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Chemistry of Materials

collected by exploiting the membrane protein extraction kit and differential centrifugation treatment.27 This method could mostly reserve the membrane components without the residue of intracellular components.20 OD solution (0.25 mg/mL) was slowly added into the dilute solution of CCCM (0.2 mg/mL) under vortexing. The obtained mixture was subject to the consecutive extrusion through a series of water-phase filters with reducing pore size (2.0 μm, 800.0 and 450.0 nm). The weight ratio (CCCM/OEI/DNA) in the feed was ultimately optimized at 5.2:2.6:1.0 to ensure the better stability and then used throughout the study (Figure S1). When coating OD with CCCM originating from human cervix carcinoma (HeLa) cells, the mean hydrodynamic diameter (Dh) of OD@CCCM NPs dropped from 300 to 200 nm (Figure S2) together with a marked decline of the zeta potential from +20 mV down to a value close to zero, as measured by dynamic light scattering (DLS), mainly caused by the charge shielding effect. Transmission electron microscopy (TEM) intuitively revealed the superficial coverage of CCCM layer (Figure 1A). Both DLS and

prerequisite of extreme sensitivity would impose rigorous troubles onto the structural stability during the circulation and the storage. Furthermore, complicated chemistry is frequently involved in the NP design to respond to the tumor microenvironment, which is certainly not desirable for practical application. Sharply different from the current chemistry methodology, this study has established a biohybrid approach to realize the role transition of pcNPs from a “lurker” during the circulation to an “attacker” upon reaching tumor sites. To this end, pcNPs are arranged to put on a biological “coat” derived from cracked cancer cell membranes (CCCM) through electrostatic interaction. The CCCM coverage is expected to shield the positive charge of pcNPs and thus suppress both the capture by biological components and the uptake by normal cells,18−20 being a “lurker” during the circulation period. Tumor cells readily agglomerate with strong adhesion to constitute solid tumors probably due to the existence of specific proteins (focal adhesion proteins, integrin, focal adhesion kinase, and RHO family proteins) on the cell membrane surface.21−25 Very recently, our lab has identified that the CCCM-coated noncharged NPs can preferentially recognize the homotypic cancer cells and actively self-home to the tumor developed from the same cancer cell lines.20 However, it is entirely unclear if this new strategy can take effect for pcNPs featured with strong interaction with cells. We hope that this unique self-recognition merit may compensate the CCCM-coating caused sacrifice of cell-uptake efficiency in homotypic cancer cells while not affecting the uptake inhibition toward heterotypic cells (i.e., macrophage and normal cells), in turn affording selective attack to cancer cells (Scheme 1). If achievable, this biohybrid Scheme 1. Illustration of Cancer-Cell-Camouflaged pcNPs (pcNP@CCCM) To Overcome Inherent Barriers of Positively Charged Nanoparticles (pcNPs) in the TumorSpecific Deliverya

Figure 1. (A) TEM images of OD and OD@CCCM. (B) SDS-PAGE protein analysis of source cancer cell membrane (I), CCCM (II), and OD@CCCM (III). (C) Hemolysis test of OEI (I), OD (II), OD@ CCCM (III). The tests in normal saline (IV) and DI water (V) served as positive and negative control, respectively. (D) Gel electrophoreses of OD@CCCM and OD upon 2 h exposure to various concentrations of heparin. (E) CLSM images of Raw264.7 cells upon 4 h incubation with OD@CCCM and OD. Green fluorescence represented the plasmids pGL-3 stained with YOYO-1. Scale bars: 20 μm. CCCM was derived from HeLa cells.

TEM manifested the effective dispersion of OD@CCCM NPs. These findings demonstrated the success of CCCM coverage by using our approach. On the basis of protein ingredient analysis, it came out that the membrane proteins belonging to the source cell membrane were well retained during the treatment (Figure 1B). A desirable drug-delivery system should possess good hemocompatibility. A hemolysis test was performed to evaluate the compatibility of OD@CCCM NPs with blood erythrocyte. Incubation of OD@CCCM in erythrocyte solution led to considerably more erythrocyte sediments than OEI and OD did after the centrifugation treatment, indicating that CCCM coating onto pcNPs could effectively inhibit the damage to erythrocyte cells (Figure 1C). Furthermore, the premature

First, pcNPs act as a “lurker“ to escape macrophage capture (A) and protein adsorption (B) as well as avoiding the non-specific uptake by normal cells (C) during the circulation period while transforming to an “attacker” to efficiently penetrate into the tumor cells upon reaching homologous tumors.

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approach may pave a facile pathway to solve the long-standing dilemma in the development of clinically applicable pcNPs particularly in the tumor-specific treatment and diagnosis. Herein, we used cationic gene nanocomplexes as the typical example of pcNPs. Lowly toxic branched oligoethylenimine (Mw = 1800 Da, OEI) was complexed with DNA at the optimal ratio of 2.6:1.0 (w/w) for transfection, providing OEI/ DNA (OD) pcNPs.26 The CCCMs from different sources were 2228

DOI: 10.1021/acs.chemmater.6b05120 Chem. Mater. 2017, 29, 2227−2231

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Chemistry of Materials DNA liberation ought to be minimized before reaching the targeted sites because naked DNAs easily undergo the degradation by serum nucleases. Negatively charged components in blood are prone to compete with DNA for binding to the cationic vectors and thus cause unwanted DNA liberation.28 In this study, heparin was used as the model of serum components for stability evaluation. As for OD pcNPs, the initially entrapped DNA completely escaped away from the gel at the heparin concentration of 2.0 mg/mL (Figure 1D). In contrast, no DNA leakages were detectable from OD@CCCM NPs even at a higher heparin concentration of 4.0 mg/mL. Antiphagocytosis ability is an important indicator reflecting the response of immune system to foreign substances. OD@ CCCM NPs were identified to exhibit negligible macrophage engulfment during 4 h coincubation with Raw264.7 macrophages, as proven by both CLSM observation and flow cytometric profiles (Figure 1E and Figure S3). Compared with the serious engulfment of parent OD pcNPs, this marked improvement indicated that the macrophage uptake can be strongly inhibited once the pcNPs were coated with CCCMs. A major challenge for the in vivo application of pcNPs arises from its susceptibility to the capture by the blood components such as proteins, which sharply reduces the bioavailability of gene payloads in the targeted sites and readily induces the risk of in vivo clearance and even thrombosis/embolism.28 The protein adsorption test was therefore carried out, using albumin from bovine serum (BSA) as the model protein (Figure S4). Compared with OD pcNPs, OD@CCCM NPs adsorbed much less BSA protein, and this decreasing tendency was represented more obviously along with the increase of CCCM amount in feed. Moreover, unlike the marked size increase over 14 days as observed for OD pcNPs in 10%-serum-containing PBS solution, the Dh of OD@CCCM NPs remained always steady under the same condition (Figure S5). It is suggested that the charge shielding by CCCM coating would largely improve the resistance against the adsorption by negatively charged serum components, which somewhat accounted for the better DNA protection of OD@CCCM NPs, the reduced damage to erythrocytes, and the lower macrophage uptake, as stated above. Taken together, all these results demonstrated that the CCCM coating would substantially improve the compatibility of pcNPs in the bloodstream. The high charge density is responsible for the easy and nonspecific uptake of pcNPs by the cells, leading to the substantial loss of carried therapeutics and the risk of side effects. In principle, it is desired that coating CCCM onto pcNPs could inhibit the nonspecific uptake by the heterotypic cells (i.e., macrophage and normal cells) while the high cell internalization efficiency (CIE) in the homotypic cancer cells remains unaffected. If feasible, this would to a great extent enable the selectively efficient uptake by the cancer cells. Cellular internalization of HeLa CCCM coated OD (OD@ HeLa) NPs was examined after 4 h of coincubation with four cell lines including HeLa, 3T3 (mouse embryonic fibroblasts), COS7 (African green monkey kidney cell), and HepG2 (human hepatocellular carcinoma cell) cells. Both CLSM observation and flow cytometric profiles showed that the CIE in three heterotypic cells indeed had a marked decrease, similar to the case in macrophage cells (Figure 2). The result is reasonable, assuredly due to the charge shielding upon CCCM coating. Of interest, the drop of surface charge close to zero after CCCM coating conversely caused much stronger uptake by homotypic HeLa cells instead of any loss of CIE. This

Figure 2. CLSM images (A) and flow cytometric data (B) of four cell lines including HeLa, 3T3, COS7, and HepG2 cells upon 4 h incubation with OD and OD@HeLa NPs. Nuclei were stained blue with Hoechst 33342 and pGL-3 was stained green with YOYO-1. Scale bars: 20 μm.

extraordinary finding proved the strong self-recognition of OD@CCCM NPs by the source cancer cells, confirming the potential of our approach to surmount the inherent barrier of pcNPs regarding the nonspecific cellular uptake. Resultantly, the mean fluorescence intensity (MFI) of OD@HeLa NPs inside HeLa cells approximated to around 3.2-fold as that obtained in heterotypic cell lines (Figure 2B). Additionally, OD@COS7 using normal cell membrane displayed apparently less uptake in both normal Raw264.7 cells and cancerous HeLa cells compared with parent OD, reconfirming the homotypic targeting capability of the CCCM-coated NPs (Figure S6). Next, the in vitro luciferase transfection of OD@HeLa NPs was conducted in HeLa, 3T3, COS7, and HepG2 cell lines to investigate the influence of selective cell uptake on the transfection. OD@HeLa NPs gave the transfection efficiency in HeLa cells at a higher level with around 10 times than OD pcNPs (Figure 3A). In sharp contrast, there appeared a significant drop of transfection efficiency in the three heterotypic cells as the result of coating OD with HeLa CCCM, assuredly ascribed to the uptake inhibition under the condition. Furthermore, the obtained CIE result reasonably explained the finding that homotypic HeLa cells displayed the highest level of protein expression among four cell lines when transfected with OD@HeLa NPs. OD@HeLa NPs mediated the transfection in HeLa cells with 17−50 times that obtained in the three heterotypic cells, reconfirming the self-recognition to homotypic cancer cells. With regard to the enhancement in homotypic cells versus in heterotypic cells, it is interesting to point out the deviation between CIE (2.5−3.3 times) and transfection efficiency (17−50 times). It seems that the coating using the source CCCM may benefit the intracellular transfection process, although the underlying mechanism is unclear. The application of pcNPs currently faces a significant challenge that the favorable in vitro performances are difficult to reproduce in vivo. As aforementioned, one of the major 2229

DOI: 10.1021/acs.chemmater.6b05120 Chem. Mater. 2017, 29, 2227−2231

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Chemistry of Materials

offer visual inspection, the transfection was conducted using pORF-LacZ gene. The transfection performance can be assessed on the basis of the expression of β-galactosidase within tumors, which showed blue color upon X-gal staining. The same conclusion was obtained. As shown in the tumor photograph (Figure 3C), a darker blue color with wider regions was evidently detectable in the tumor treated with OD@UMSCC-7 NPs compared with other two controls using either OD or OD@HeLa NPs. The observation on the paraffin-embedded section further provided evidence regarding the β-galactosidase expression inside the depth of tumors (Figure 3D). These results agreed fairly well with the in vivo quantitative data acquired by using pGL-3 plasmid as reporter gene (Figure 3E). The luciferase expression level of OD@UM-SCC-7 NPs in the tumor was approximately 26 and 10 times higher than that of OD and OD@HeLa, respectively. As we expected, the highly efficient protein expression mediated by OD@CCCM NPs was acquired in the homologous tumor developed from the same cancer cell lines, consistent with the in vitro outcome obtained at cellular levels. Noticeably, OD@HeLa NPs exhibited much better transfection performance in the heterologous tumor than OD, as contrary to the in vitro outcome that the coating with CCCM would reduce the transfection efficiency in the heterotypic cells. The result can find the explanation based on the in vitro compatibility experiments. It is suggested that post intravenous injection, OD@CCCM could act as a “lurker” that efficiently escapes the “capture” of foreign substances commonly occurring for pcNPs, such as the adsorption by blood components and the macrophage uptake. Compared with OD pcNPs, OD@CCCM thus afforded the largely enhanced bioavailability of carried DNAs and better transfection in the heterologous tumor. From another perspective, this finding reconfirmed the advantage of using CCCM to coat pcNPs for in vivo application. In summary, we put forward a biohybrid strategy instead of the chemistry methodology to render pcNPs the ability of role transition from a “lurker” to an “attacker” upon reaching tumor sites. CCCM coating to pcNPs could effectively depress the adsorption by biological components, the macrophage capture, and the nonspecific uptake by normal cells. On the other hand, the “biological identity” ascribed to CCCM coating makes it achievable for the cancer cell self-recognition and the selftargeting to the homologous tumor developed from the source cancer cells. The experiments using CCCMs derived from different cancer cells suggest the universality of this biohybrid strategy. This strategy may pave a facile way to address the commonly existing limitations for in vivo application of pcNPs.

Figure 3. (A) In vitro serum-conditioned transfection in four cell lines mediated by OD and OD@HeLa NPs. (B) Representative fluorescence micrographs of tumor frozen sections obtained at 24 h after injection of pEGFP-containing NPs including OD, OD@HeLa, and OD@UM-SCC-7. Magnification 200×. (C) Images of X-gal stained tumors separated at 2 day and paraffin-embedded section (D) at 24 h post intravenous injection of pORF-LacZ-containing NPs including OD, OD@HeLa, and OD@UM-SCC-7. (E) Luciferase expression in UM-SCC-7 tumors after treatment with different pGL-3containing NPs. *p < 0.05 and **p < 0.01 and ***p < 0.001 were determined by a Student’s t test.

causes is the effective capture by the oppositely charged blood components, leading to the substantial loss of carried therapeutics. Hence, the performance of in vitro serumconditioned transfection has been acknowledged as an important index to prejudge the in vivo transfection outcome.26 The obtained data showed that the transfection mediated with OD@HeLa NPs always remained at a high level with almost no changes as serum concentration increases up to 30% (Figure S7). Under the serum-containing condition, the CCCMcoating-caused transfection improvement (∼100 fold) in homotypic HeLa cells was represented more obviously compared with that (∼10 fold) in serum-free medium. This is mainly due to the inhibited adsorption by serum components upon CCCM coating. The transfection using plasmid EGFPC1 encoding green fluorescent protein (GFP) as reporter gene further provided visual examination over the influence upon serum addition (Figure S8). Similarly, a considerably higher level of GFP expression was detected for the transfection with OD@HeLa CCCM NPs in HeLa cells than the control using OD, whereas HeLa CCCM coating resulted in a reduction or no significant changes of fluorescence intensity in the heterotypic cells. The finding agreed well with the quantitative analyses of luciferase transfection. Encouraged by the in vitro results, we next roughly evaluate the potential of OD@CCCM NPs for the in vivo application. OD and OD@CCCM NPs containing different types of plasmids (pEGFP, pORF-LacZ, and pGL-3) were intravenously administrated into the BALB/c nude mice bearing a UM-SCC7 cell (human squamous carcinoma cell lines developed at the University of Michigan) xenograft tumor. The GFP expression treated with pEGFP-containing OD@UM-SCC-7 NPs in the homologous tumor was much stronger than that with OD@ HeLa NPs or OD pcNPs, as reflected from the brightness of green fluorescence in tumor frozen sections (Figure 3B). To

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b05120. Preparation, characterization of OD@CCCM NPs, and other experimental details (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jun Feng: 0000-0002-1725-140X Xianzheng Zhang: 0000-0001-6242-6005 2230

DOI: 10.1021/acs.chemmater.6b05120 Chem. Mater. 2017, 29, 2227−2231

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Chemistry of Materials Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (Nos. 21374085, 21174110, and 2011CB606202) and Natural Science Foundation of Hubei Province of China (2014CFB697).

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ABBREVIATIONS

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REFERENCES

CCCM, cracked cancer cell membrane; pcNPs, positively charged nanoparticles; OD, OEI/DNA complex; CIE, cell internalization efficiency

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DOI: 10.1021/acs.chemmater.6b05120 Chem. Mater. 2017, 29, 2227−2231