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PEI-NIR Heptamethine Cyanine Nanotheranostics for Tumor Targeted Gene Delivery Eduardo De los Reyes-Berbel, Rafael Salto-Gonzalez, Mariano Ortega-Muñoz, Francisco Jose Reche-Perez, Ana Belén Jódar-Reyes, Fernando HernándezMateo, Maria Dolores Giron-Gonzalez, and Francisco Santoyo-González Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00262 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 30, 2018
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Bioconjugate Chemistry
PEI-NIR Heptamethine Cyanine Nanotheranostics for Tumor Targeted Gene Delivery Eduardo De los Reyes-Berbel,a Rafael Salto-Gonzalez,b Mariano Ortega-Muñoz,a Francisco Jose Reche-Perez,b Ana Belen Jodar Reyes,c Fernando Hernandez-Mateo,a Maria Dolores Giron-Gonzalez,b,* and Francisco Santoyo-Gonzaleza,* a
Department of Organic Chemistry, Biotechnology Institute, Faculty of Sciences, University of Granada, 18071-Granada, Spain b
Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, 18071 Granada, Spain
c
Biocolloid and Fluid Physics Group, Department of Applied Physics, Faculty of Sciences, University of Granada, 18071-Granada, Spain
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ABSTRACT Polymer-based nanotheranostics are appealing tools for cancer treatment and diagnosis in the fast-growing field of nanomedicine. A novel straightforward preparation of novel engineered PEI-based nanotheranostics incorporating NIR fluorescence heptamethine cyanine dyes (NIRFHC) to enable them with tumor targeted gene delivery capabilities is reported. Branched PEI2kDa (b2kPEI) is conjugated with IR-780 and IR-783 dyes by both covalent and non-covalent simple preparative methodologies varying their stoichiometry ratio. The as-prepared set of PEINIR-HC nanocarriers are essayed in vitro an in vivo to evaluate their gene transfection efficiency, cellular uptake, cytotoxicity, internalization and trafficking mechanisms, subcellular distribution and tumor specific gene delivery. The results show the validity of the approach particularly for one of the covalent IR783-b2kPEI conjugates that exhibit an enhanced tumor uptake, probably mediated by organic anion transporting peptides, and favorable intracellular transport to the nucleus. The compound behaves as an efficient nanotheranostic transfection agent in NSG mice bearing melanoma G361 xenographs with concomitant imaging signal and gene concentration in the targeted tumor. By this way, advanced nanotheranostics with multifunctional capabilities (gene delivery, tumor-specific targeting and NIR fluorescence imaging) are generated in which the NIRF-HC dye component accounts for simultaneous targeting and diagnostic avoiding additional incorporation of additional tumor-specific targeting bioligands.
KEYWORDS: gene delivery; tumor-specific targeting; polyethylenimine; heptamethine cyanine dyes; polymeric nanocarriers
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INTRODUCTION Polymer-based nanosystems are promising non-viral gene delivery carriers. For this endeavor, polymers are attractive not only because they are safer than viruses but also cost-effective and easier to engineering and tailoring than others gene delivery vehicles.1-3 Polyethylenimines (PEIs) are cationic polymers of common choice for this endeavor due to their inherent properties for the efficient delivery of nucleic acids.4-6 PEI-based nanocarriers electrostatically complex with negative charged DNA to form polyplexes that prevent charge repulsion of the genetic material with negatively charged cell membrane and protect from nucleases degradation. PEI-based gene vectors also offer distinctive characteristics that enables them for targeted gene delivery,7-9 a key issue to ensure site-specific gene accumulation and tumor-specific transfection in cancer therapy.10-12 The ready supply of nucleophilic amines in PEI offers unique possibilities that can be exploited on either passive and active targeting delivery strategies.4 Covalent bio-functionalization of PEI is a well-studied method for their rational engineering in active targeting strategies aimed to incorporate bioligands that are discriminatorily recognized and specifically bound by expressed or over-expressed cell surface receptors.13, 14 Transferrin, folate, specific carbohydrates, antibodies, proteins and peptides, and have been extensively utilized as targeting moieties for selective PEI-based polyplexes delivery to tumor cells.4 In parallel with the demand of targeted polymeric nanosystems for gene therapy, there is an increased interest in polymeric-based nanotheranostics as therapeutic agents possessing concomitant imaging capabilities.15 These systems are intended for image-guided therapies that monitor distribution and release of drugs or genes and simultaneously allow evaluation of the therapeutic efficacy non-invasively in real time.16-18The outstanding biocompatibility, biodegradability, structural versatility19 and possibilities for the incorporation of targeting ligands to further enhance their specific delivery to selected targets are also attractive characteristics of these nanoplatforms.
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In the case of image-guided gene delivery, fluorescence is the most common optical imaging technology used to track the gene transfection/delivery events. The use of fluorescence enable a better understanding and improvement of the fate of the gene and/or the delivery vehicles through the different physiological barriers, either in vitro or in vivo. Different fluorescent-based polyplexes have been prepared as nanotheranostics either by the concurrent or independent staining/labelling of the polymeric carrier20,
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and/or the genetic material,22 or by the use of
fluorescent particles, usually quantum dots (QDs), coated with a cationic polymer.23, 24 Among the fluorescence technologies, near-infrared fluorescence (NIRF) imaging (700-1000 nm wavelength range) has attracted great attention in theranostics owed to their intrinsic advantages: use of nonionizing radiation, low autofluorescence and absorption from organisms and tissues that minimize background interference, non-invasive and sensitive optical imaging, and improve tissue depth penetration.25 Consequently, the development of NIRF probes has become a major focus on research being polymethine cyanine dyes the most widely used NIRF. Among them Cy5, Cy5.5, Cy7 and their derivatives, and also indocyanine green are the most prevalent employed NIRF probes.26 Recently, some NIRF heptamethine cyanine dyes (NIRF-HC) (namely IR-780, IR-783, IR-808 and MHI-148) have demonstrated to be promising anticancer theranostic agents27 due to the inherent cancer-specific targeting capabilities of their native forms absent in others NIRF probes. NIRF-HC dyes show no accumulation in normal cells but a preferential accumulation and retention in different types of cancer cells. This fact enables cancer-specific targeting avoiding chemical conjugation of additional tumor-specific targeting bioligands. In this context, pairing a PEI scaffold, as a polycationic vehicle, and a NIRF-HC probe, as a detection agent for imaging and a targeting motif, is an attractive tandem for the development of multifunctional advanced targeted nanotheranostics gene vectors. The strategy has been scarcely used28-33 for IR-820 dye through its covalent linkage or electrostatic co-delivery in the development of organ-selective (brain29-31 and lung32) or tumor-selective33 gene carriers with 4 ACS Paragon Plus Environment
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selective transport mediated by conjugated bioligands (fatty acids or peptides) but not by NIRFHC dyes.
With this background, we hypothesize that efficient theranostic polymeric
nanoplatforms for tumor targeted gene delivery could be engineered by simple conjugation of PEI 2kDa (b2kPEI) with NIRF-HC dyes possessing native cancer-specific abilities, namely IR780 and IR-783 (PEI-NIRF-HC nanocarriers) without the need of further targeting ligands. We report herein the straightforward preparation of such nanomaterials as well as in vitro and in vivo studies that corroborate their capabilities as advanced nanotheranostics with multifunctional capabilities: gene delivery, tumor-specific targeting and NIR fluorescence imaging. RESULTS AND DISCUSSION 1. Design and synthesis of NIRF-HC-PEI nanocarriers With the aim to prepare efficient NIRF-HC-based nanotheranostics with targeting capabilities, we undertook the conjugation of NIRF-HC dyes with PEI because of the synthetic simplicity of this strategy. We selected branched PEI-2kDa (b2kPEI) among the commercial polyethylenimine derivatives because of its favorable low cytotoxic profile. For PEI/DNA polyplexes, the transfection efficiency and cytotoxicity are mainly dependent on the PEI physicochemical properties having been reported that low molecular weight PEIs are those that exhibit lower cytotoxicity.34, 35 Likewise, we selected IR-780 and IR-783 as prototypical multifunctional NIRFHC dyes because of their proven NIR imaging and tumor targeting capabilities.26, 27 IR-780 and IR-783 exhibit appealing optical properties, namely, increased photostability and fluorescence quantum yield, and low photobleaching with simultaneous low dye aggregation. However, their structural differences (alkyl chains for IR-780 and alkyl sulfonates for IR-783) are known to determine different solubility properties (liposolubility vs hydrosolubility, respectively) and toxicity profiles (favorable for IR-783), parameters that would be evaluated to establish their influence in the transfection efficiency.
The rigid chloro-cyclohexenyl ring of IR-780 and IR-
783 is normally the reactive site used for their chemical modification and/or conjugation.36 Taking into account that large Stokes shifts and stronger fluorescence are characteristic of NIRF5 ACS Paragon Plus Environment
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HC dyes containing C-N bonds,37, 38 we decided to use the piperazine derivative of IR-780 and IR-783 (compounds IR780p and IR783p) instead of the native dyes. Compound IR783p has been previously prepared by straightforward displacement of the central chlorine atom by piperazine. A high chemical stability for this derivative has been also reported.39 Following this methodology, compound IR780p was prepared by us in a similar manner (Scheme 1). R2 N Cl
N
a
N+ R1
N R1
IR780 R1= (CH2)2CH3 IR783 R1 = (CH2)4SO3-Na+
N+ R1 b b
N
O S O IR780pVS IR783pVS
+ NH2
N
N H H2N
N R1
IR780p R1= (CH2)2CH3, R2 = H IR783p R1 = (CH2)4SO3-Na+, R2 = H IR780pVS R1= (CH2)2CH3, R2 = (CH2)2SO2CH=CH2 IR783pVS R1 = (CH2)4SO3-Na+, R2 = (CH2)2SO2CH=CH2
N N H n
c NH2
NH2
b2kPEI
N
N H H2N
N H n
N H IR780pVS-b2kPEI IR783pVS-b2kPEI
O S O
Scheme 1. Synthesis of NIRF-b2KPEI covalent nanocarriers. Reagents and conditions: a) piperazine, 80 ºC, 4 h, DMF; b) DVS, Et3N, 16 h, RT, CH2Cl2-MeOH; c) MeOH, 16 h, RT To anchor IR780p and IR783p to b2kPEI and on the basis of our previous experience on the (bio)applications of the clickable vinyl sulfone (VS) function,40 we decided to transform these amino cyanines into the corresponding vinyl sulfone derivatives IR780pVS and IR783pVS with the aim to use vinyl sulfone click-chemistry as an efficient conjugation methodology. Remarkable advantages of the Michael-type addition of vinyl sulfones with nucleophiles as click counterparts are their robustness, simplicity, elevated water stability of both the VS group and the linkage formed upon conjugation, and the absence of by-products. In fact, the labelling of biomolecules with VS-containing NIRF polymethine cyanine dyes has been previously reported.41, 42 Conversely to the described multistep strategies for accessing such compounds, the clickable VS group was introduced in the present case by simple treatment of IR780p and IR783p with divinylsulfone (DVS) (Scheme 1). Chemical conjugation of the resulting VS 6 ACS Paragon Plus Environment
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functionalized probes, IR780pVS and IR783pVS, to b2kPEI is easily carried out by direct mixture of those reagents giving conjugates IR780pVS-b2kPEI and IR783pVS-b2kPEI in good yields (Scheme 1). Successful coupling was confirmed by absence on those conjugates of the characteristic signals of VS group in NMR. With the aim to develop simple preparative protocols, we also assayed the non-covalent self-assembling of IR780p and IR783p with b2kPEI. Formation of the corresponding NIRF-HC@b2kPEI aggregates was easily performed by direct mixture of the reagents and evaporation. The aggregation is particularly facilitated for IR783p because of the favorable electrostatic interactions between the anionic sulfonate groups of their hydrophilic alkyl pendants and polycationic b2kPEI (see Figure S1). A similar co-delivery noncovalent strategy has been previously reported in the design of engineered indocyanine greenloaded carriers43 and related polyionic-based carriers.44, 45 In both strategies (covalent and non-covalent conjugation) two NIRF-HC:b2kPEI stoichiometry ratios (namely, 1:1 and 2:1) were used to address the influence of this parameter in the transfection efficiency. In addition, all NIRF-HC-PEI nanocarriers conserve the photophysical properties of the native NIRF-HC dyes that enables them as NIR fluorescent theranostic reporters (Figure S2). 2. In vitro and in vivo transfection capabilities of NIRF-HC-PEI nanocarriers in non-tumor cells 2.1. In vitro gene delivery capabilities of NIRF-HC-PEI nanocarriers To evaluate in vitro the capabilities of the as-prepared NIRF-HC-PEI derivatives to bind and compact plasmid DNA (pDNA) and protect it from DNA degradation they were combined with plasmid pEGFP-N3, which encodes for the green fluorescent protein, at different N/P ratios (0.1 to 1.75). The DNA binding and release of the resulting NIRF-HC-PEI/pDNA polyplexes were evaluated and compared with that of b2kPEI/pDNA polyplex by a gel retardation assay (Figure S3). The intensity of the DNA bands was normalized and plotted against the N/P ratio used. (Figure 1A). Data indicated that incorporation of a NIRF-HC dye to b2KPEI translates in increased DNA binding affinity of the nanocarriers. The increase is higher for IR780-based 7 ACS Paragon Plus Environment
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nanocarriers compared with those derived from IR783, observation that can be ascribed to the higher hydrophobicity of the IR780 dye and the presence of negatively charged sulphonic groups in these last vectors. Interestingly, while the conjugation method (covalent or non-covalent) has no effects on the DNA binding capability of IR780-based compounds, the IR783 non-covalent nanocarriers exhibit a higher binding capability compared with their covalent counterparts.
Figure 1. Gen delivery capabilities of NIRF-HC-PEI/pEGFP-N3 polyplexes. (A) Gel electrophoresis shift assay: Binding and release calculated by quantification of the intensity of plasmid bands (mean values of relative percentage for three independent experiments). (B) DNase I protection experiments: Relative intensity of relaxed and supercoiled electrophoretic plasmid bands treated with DNase I (100 value correspond to untreated pEGFP-N3). Results expressed as means ± SEM (n=6). * p
