Targeted Gene Delivery to Macrophages by Biodegradable Star

Letter www.acsami.org

Targeted Gene Delivery to Macrophages by Biodegradable StarShaped Polymers Yajie Zhang,† Yafeng Wang,‡ Chi Zhang,† Jin Wang,§ Dejing Pan,‡ Jianghuai Liu,*,‡ and Fude Feng*,† †

Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China ‡ MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center of Nanjing University and National Resource Center for Mutant Mice, Nanjing 210061, P. R. China § Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, United States S Supporting Information *

ABSTRACT: In this report, two biodegradable star-shaped polyasparamide derivatives and four analogues modified with either mannose or folic acid moiety for preferential targeting of a difficult-to-transfect immune cell type, i.e., macrophage, have been synthesized. Each of the prepared star polymers complexes with plasmid DNA to form nanosized particles featuring a core−shell-like morphology. Mannose or folate functionalized star polymers can greatly improve the transfection performance on a macrophage cell line RAW 264.7. As a result, a combination of targeting ligand modification and topological structures of gene carriers is a promising strategy for immune cells-based gene therapy. KEYWORDS: targeted gene delivery, core−shell, star polymer, ring opening polymerization, macrophage

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patients to avoid rejection reaction of xenograft in the next treatment procedures. After ex vivo transfection, the TAMs maybe propagated in culture, inoculated back to tumor, and used to modify the tumor microenvironment and to enhance antitumor immunity. In addition to gene manipulation as a single treatment regimen, a combination of nucleic acids therapy and immunotherapy has the potential to serve as a powerful tool for treatment of many diseases in clinic, especially cancers and inflammatory diseases identified with macrophageinfiltrated tissues.10,11 Nevertheless, one of the most challenging part of nucleic acids therapy is to safely deliver therapeutic gene(s) to targeted tissues and cells that need precise regulation in gene functioning. Indeed, the naked nucleic acids can be hardly targeted to selected destinations because of the presence of a number of physiological barriers such as nuclease attack, inefficient cellular internalization, and lack of targeting property.12 Up to now, to meet the urgent demand of immune cells-targeted gene therapy, virus-based gene carriers, such as adeno-associated virus (AAV), are the most widely used vector systems in research and clinical investigations due to its high efficiency in transduction of immune cell types. However, safety concerns remain the major hurdle for the clinical use of viral

acrophages, a type of phagocytes, are present in significant numbers in almost all tissues of human body and play vital roles in the immune system in response to environmental cues.1 Under normal conditions, macrophages self-maintain in most of the tissues. Tissue injuries by insults from microbial or self-origins cause additional macrophage precursors recruited to the damaged site and subsequently activated. Determined by the natures of tissue injury and associated inflammatory milieu, activated macrophages can exhibit two main phenotypes, termed M1 (classical activation) and M2 (alternative activation). M1 macrophages produce a significant amount of tumor necrosis factor (TNF) and interleukins (IL-12, IL-23) to elicit Th1/Th17 (T helper lymphocyte 1 and 17) immunity and amplify inflammatory responses. M2 macrophages have an immunosuppressive function and contribute to tissue repair and remodeling, direct pathogen clearance, alleviating inflammation as well as tumor progression.1−3 The two phenotypes are important in maintaining homeostasis of our body and modulating inflammatory response.4,5 Tumor-associated macrophages (TAMs), as a typical example of M2 phenotype, are highly relevant in cancers and have been shown to promote the malignant disease via facilitating tumor growth, angiogenesis and metastasis, while modulating inflammation and driving immunosuppression.6−8 Thus, a proposed method for cancer treatment is to switch the phenotype of TAMs from M2 to M1 by nucleic acids therapy.9 The therapeutic gene can be inserted into TAMs isolated from © XXXX American Chemical Society

Special Issue: Applied Materials and Interfaces in China Received: August 31, 2015 Accepted: September 30, 2015

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DOI: 10.1021/acsami.5b08119 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Design of Biodegradable Star-Polymers As Gene Carriers for Targeted Nucleic Acid Delivery to Macrophagesa

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(a) Star polymers that are modified with target ligands assemble with DNA encoding gene of interest to result in core-shell nanoparticles. Condensed DNA along with polymer chains neutralizing negative charges of DNA resides in the core. The nano-thick shells composed of polymers shield DNA from environmental contact. Physico-chemical properties of the core-shell nanoparticles basically rely on the roles of charges and ligands distributed on the shell surface. (b) Core−shell nanoparticles undergo receptor-mediated intracellular internalization and proton sponge effectmediated endosomal escape process. As a result, a proportion of payload DNA is delivered to nucleus where gene expression machines are running. (c) Synthetic route of SP1-DET and SP2-DET. The structures resulting from racemization during aminolysis are shown.

vectors.13 As an alternative, nonviral gene carriers are attractive thanks to the advantages including broad availability, economic cost-effectiveness, low cytotoxicity, friendly operation, and diversified functionalization. Recently, great efforts have been made in improving transfection performance by rationally designing chemical architectures of synthetic gene carriers, cationic polymers in particular.14−17 High in vitro delivery efficiency has been achieved by utilizing branched and star-shaped hydrophilic polymers.18−20 Furthermore, in a previous study, an eight-arm cationic poly(aspartic acid) derivative with capability of proton sponge effect and nonenzymatic degradability of polymer backbones was found to prevail over its linear counterparts and commercial polyethylenimine (PEI, a gold standard transfection reagent) in both in vitro and in vivo transfection of lung cancer cells or lung tissues with EGFP plasmid.21 These findings inspired us to extensively apply poly(aspartic acid)based star polymers to transfect immune cells, such as macrophages.

Macrophages have been proven difficult to transfect attributed to significant intracellular ROS production that is detrimental to plasmid integrity.22 Targeted gene delivery system has been found beneficial in increasing cellular internalization and thereby improving transfection efficiency via introduction of target ligands that are recognized by receptors overexpressed in the cell surface.23 Mahor and coworkers reported targeted gene delivery to RAW 264.7 macrophages using mannosylated polyethylenimine-hyaluronan nanohybrids as gene carrier with transfection efficiency up to ∼7.5% examined by flow cytometry analysis, which demonstrates that macrophage transfection can be facilitated by mannosylation but remains to be improved.24 In this work, biodegradable star cationic polymers were explored for targeted gene delivery to macrophage RAW 264.7 cells after low degree of mannosylation and folate functionalization for effective endocytosis mediated by mannose- and folic acid receptors respectively that are known to be overexpressed in RAW 264.7 (Scheme 1a, c).10,24 The cationic B


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Figure 1. GPC traces of the star polymers: (a) SP1 and (b) SP2. 1H NMR spectra (600 MHz, 298 K) of (c) SP2 in DMSO-d6 and (d) SP2-DET in D2O.

originates from two factors, one with the difference in hydrodynamic volumes of PBLA and polystyrene, and another one with the difference in structural architecture of multiarm PBLA and linear polystyrene.25 Additional attempt to acquire Mn was carried out based on NMR experiments which are known to be favorable in estimating molecular weight of macromolecules.26 DPNMR, defined as the number of repeating unit of star polymer, was calculated from the integral of proton resonance of the benzyl (−CH2C6H5) and that of the core of star polymer (PEI or DAB-Am-4) as well as the side chain (−CH2COOCH2C6H5), obtained as 126 and 98 for SP1 and SP2, respectively. The 1H NMR spectrum of SP2 was shown in Figure 1c and the comparison of Mn,GPC and Mn,NMR is summarized in Table S1. Cationic star polymers were obtained by facile aminolysis of benzyl groups on the side chains of neutral precursors in the presence of excess diethylene triamine (DET) in N-methyl-2pyrrolidone (NMP) and subsequent careful protonation on each primary amine group freshly generated. The aminolysis conversion percentage is analyzed by 1H NMR (Table S1) and estimated to be nearly 100%. Kataoka group’s and our previous studies have confirmed that DET-modified polyaspartamides in aqueous solution undergo nonenzymatic hydrolysis of backbones facilitated by elevating temperature and pHs, ensuring degradable property in physiological condition.27,28 The nonprotonated secondary amines on the side chains of cationic polymers are available for a well-known proton sponge effect that favors endosomal escape of intracellularly internalized nanoparticles.

polymers were derived from PBLAs aliphatic polyaspartamides Polyethylenimine-Poly(β-benzyl-L-aspartate) (SP1) and Polypropylenimine tetramine dendrimer-Poly(β-benzyl-L-aspartate) (SP2) that were obtained through ring-opening polymerization (ROP) of β-benzyl-L-aspartate N-carboxyanhydride (BLANCA). The amino groups of branched PEI (1.8 kDa) or polypropylenimine tetramine dendrimer (DAB-Am-4) are effective metal catalyst-free ROP initiators to allow for chaingrowth polymerization under mild conditions (Scheme 1c), with SP1 being more structurally branched. The theoretical number-average molecular weight of SP1 and SP2 is roughly calculated based on the following equation M n,th = nm /n iMRU + M i

Where nm/ni is the molar ratio of monomer and initiator applied in the ROP (nm/ni = 240). MRU and Mi are molecular weights of each repeat unit and initiator, respectively. Determination of molecular weights was carried out by gel permeation chromatography (GPC) employing a refractive index detector with linear polystyrene as calibration standard. Unimodal GPC curves (Figure 1a, b) featured with a narrow molecular weight distribution (∼1.2 in PDIs) demonstrates well-controlled polymerization. In comparison to symmetrical curvature of SP2, the GPC trace of SP1 displays slightly asymmetry and broadened, attributed to the ununiformity of chemical structure of initiator branched-PEI (Mn 1.8 kDa, PDI ∼1.1). The GPC measurements give a Mn,GPC of 70.7 kDa or a DPGPC of ca. 332 for SP1, and a Mn,GPC of 49.8 kDa or a DPGPC of ca. 242 for SP2. The deviation from theoretical values mainly C


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polymers are very stable during storage in the absence of DNA over 7 days (data not shown). Generally, plasmid polyplexes have a nanosized globular shape that can be imaged by TEM technique using uranyl acetate (UA) stained samples. The images obtained by conventional TEM (Figure S3, Supporting Information) which is incompetent to visualize sub-10 nm fine structures are very common to reported nanoparticles such as PEI/ plasmid polyplex.29,30 Interestingly, core/shell like morphologies of polyplex nanoparticles (Figure 3) are successfully

In an attempt to optimize the performance of star cationic polymers for gene delivery to macrophage RAW 264.7, investigation of functionalization with widely used target ligands mannose and folic acid were performed. Folic acid was coupled to star cationic polymers at a feed molar ratio of 1:40 (folic acid: DET moiety) by employing an EDC/NHS activation strategy, and the folate products (SP1-DET-F and SP2-DET-F) were identified by 1H NMR showing proton resonances assigned to covalently conjugated folate group without sign of free folic acid (Figure 2). Similarly,

Figure 3. HR-TEM images of polyplex nanoparticles prepared by pEGFP with the following polymers at N/P 10: (a) SP1-DET, (b) SP2-DET, (c) SP2-DET-F, (d) SP2-DET-M, (e) SP1-DET-F, and (f) SP1-DET-M.

Figure 2. 1 H NMR spectra of folic acid (b) and (g) 4isothiocynatohpenyl α-D-mannopyranoside in DMSO-d6, and (a) SP1-DET-F, (c) SP2-DET-F, (d) SP1-DET, (e) SP2-DET, (f) SP1-DET-M, and (h) SP2-DET-M in D2O.

captured everywhere as seen in all of the images acquired by high resolution TEM (HR-TEM), which provides insight into the structure of nanoparticles that have been neglected. Undoubtedly, the dense cores with diameters in the 50−80 nm ranges indicate condensed plasmid DNA, which strongly absorbs UA by phosphonate bonding. The cores are coated by less dense but homogeneous shells with thickness in the 5−20 nm ranges. Both of the outer and inner shell boundaries are clearly legible. Plasmid DNA is not possible to reside in shells. It is widely accepted that polyplex particles are surrounded by excess free cationic polymers according to that the resulting particles have positively charged surface, as evidenced by the ζpotential measurements of star polymer/plasmid complexes (Table S2). Thus, it is revealed by the HR-TEM data that the uniformed nanothick shells entrapping condensed DNA are composed of free cationic polymers. This point is important that existence of thick polymeric shells ensures antiaggregation property of polyplex particles and protection of enclosed DNA against nuclease attack. To evaluate gene delivery capability of the six star polymers, we performed in vitro transfection assays on RAW 264.7 macrophage cells. At 36 h post transfection with pEGFP, cells expressing EGFP were imaged using a fluorescence microscope (Figure S3). Quantitative analysis was available by flow cytometry assay using jetPEI and Lipofectamine 2000 as positive controls (Figure 4a and Figure S4). In each case,

mannosylated products (SP1-DET-M and SP2-DET-M) were prepared using an isocyanate activation approach. The low degree (up to 2.5%) of ligand substituents is expected to have negligible interruption with polyionic complex formation between ligand-modified polymers and plasmid DNA. Agarose gel electrophoresis was run to confirm complete complexation of EGFP plasmid (pEGFP) with each of the six star cationic polymers through entropy-driven ionic interaction. As shown in Figure S2, plasmid migration in gel was totally retarded at N/P ratio of 5, indicative of full neutralization of anionic plasmid by polycations no matter whether ligand substituents were introduced (see the Supporting Information for details). After these polyplexes were stored at 4 °C for 48 h, another run of agarose gel indicated appearance of bands assigned to free plasmid without DNA fragmentation, suggesting that a fraction of plasmid was liberated from polyplexes as a result of decreased binding affinity of polycations toward DNA. The observation of DNA liberation agrees well with the degradability of polyaspartamides. It is speculated that DNA-bound DET-modified polyaspartamide undergoes faster pH-dependent hydrolysis process than its free form, associated with DNA-generated microenvironment where DET chains reside. It should be noted that chilled free D


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Figure 4. Transfection results on RAW264.7 cells obtained by (a, b) flow cytometry analysis and (c) Western blot analysis.

chemical structures of star polymers is under study to substantially boost the overall immune cell-targeted gene delivery efficiency.

transfection efficiency is improved by functionalization of the star polymers with either mannose or folic acid. Interestingly, mannosylation has a more profound effect on star polymer SP1-DET than the folate modification. However, this effect is reversed on star polymer SP2-DET (Figure 4b). In addition, when modified with either mannose or folic acid ligand, the four-arm star polymer outperforms the highly branching counterpart. These results reflect that the effectiveness of receptor-mediated endocytosis pathway is influenced by chemical architectures of gene carrier and the overall transfection performance is not simply increasing with larger branching degree. Among the six star polymers, SP2-DET-F exhibits the most significant transfection with efficiency of 8.3%, in good agreement with Western blotting results that only SP2-DET-F displays detectable GFP expression (Figure 4c). In conclusion, six biodegradable cationic star polyaspartamides were synthesized by clean ROP reaction and characterized by GPC and 1H NMR. These polymers are able to condense plasmid DNA into nanosized globular particles. For the first time, the particles were visualized to have a distinct core−shell like morphology using HR-TEM. The role of the free polymer chain-rich shells deserves an indepth investigation on how to mechanistically understand their contribution to cellular internalization, endosome interruption, and transfection performance from a general view. Star polymers without the targeting ligand modification have poor in vitro macrophage transfection capability. Taking advantage of receptor-mediated internalization, gene delivery efficiency is significantly improved with folate or mannose functionalization on the side chains of star polymers. Additional optimization on

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08119. Experimental details and figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We’re grateful to Prof. Wei Wang (Nanjing University) for help with ζ-potential measurements and Dr. Wei Xia (Nanjing University) for help with art design. We thank the National Natural Science Foundation of China (Grant 21474046), 1000 Young Talent Program, Collaborative Innovation Center of Chemistry for Life Sciences, and the Program for Changjiang Scholars and Innovative Research Team in University for financial support.

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Targeted Gene Delivery to Macrophages by Biodegradable Star

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