Synthesis and evaluation of the targeted binding of RGD-containing PEGylated-PEI/DNA polyplex micelles as radiotracers for a tumor-targeting imaging probe

Abstract

Site-specific labeling of molecular imaging probes necessitates the topical administration of medications during the development of homogeneous tracers. Hence, receptor-mediated gene transfer is believed to be of enormous significance in the clinical translation of a promising gene delivery technique. Plasmid DNA (pEGFP) and polycations produce polyplexes, which can be proficient probes for molecular imaging when accompanied by a gamma emitter. Therefore, this study describes the physico-biological characterization of a radiotracer for tumor imaging in a HeLa tumor-bearing mouse model. Polyplex micelles were formed with pEGFP and Arg-Gly-Asp (RGD) peptide-modified poly(ethylene glycol)-grafted polyethylenimine (E[c(RGDyK)]₂-PEG-g-PEI) and were labeled with ^99mTc for the in vivo study. The various PEG-g-PEIs prepared by controlling the PEG-to-PEI ratios were confirmed by ¹H-NMR. The sizes and zeta potentials of the PEG-g-PEI/DNA polyplexes were 90–135 nm and 40–50 mV, respectively. The biophysical characterization of pEGFP in polyplexes was evaluated via various methods, including determination of the condensation efficiency of the polymers and the biodistribution, in vitro stability, in vivo application, and kinetics of the radiolabeled polyplexes. These characteristics were studied as a function of time using 3D-SPECT/CT images and by end-point scintillation counting. The polyplex of PEG-g-PEI/DNA fabricated with a PEG/PEI ratio of 10 : 1 and N/P = 1, i.e., PP10/D, exhibited the lowest cytotoxicity and the highest transfection efficiency. The cyclic-RGD peptide-modified polyplex PEG-g-PEI/DNA (RPP10/D) had significantly higher binding affinity and transfection efficiency than the non-targeting PP10/D did. ^99mTc radio-labeled PP10/D and RPP10/D were prepared with high radiolabeling efficiency of greater than 95% and radiochemical stability above 80%, both in saline and in rat plasma, when stored for 24 h and were evaluated for their tumor-targeting capability and biodistribution. Through in vivo SPECT/CT images, it was determined that RPP10/D-^99mTc presented higher uptake in the tumor than PP10/D-^99mTc at all of the post-injection times studied. We found that the two tracers of radioactive complexes mainly accumulated in the liver, spleen and kidneys at 24 h after intravenous injection in female BALB/c nude mice bearing subcutaneous HeLa tumors. The accumulation of the site-specifically labeled RPP10/D-^99mTc was lower in liver, kidney and spleen compared with non-targeting PP10/D-^99mTc.

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Introduction

The introduction of a genetic substance into human diseased cells is a severe bottleneck in the progress of gene delivery for many clinical applications.^1,2 An appropriate gene delivery system is a prerequisite for cellular uptake; the transfer of large hydrophilic and charged molecules through the cellular membrane of living cells is hampered, and the undesirable extracellular circumstances provide vast opportunities to degrade DNA.³ Although the efficiencies of DNA condensation and decondensation are of significant importance in gene delivery progress, a vector's biocompatibility is also an essential factor.⁴ Thus, the formation of DNA condensates has been induced by the addition of non-viral carriers in vitro such as metal cations, cationic polymers, liposomes, peptides and polyamines.^5–7 Synthetic cationic polymers have been widely studied as a major group of non-viral carriers.⁸ Hitherto, polyethylenimine (PEI) has been considered the most potent non-viral gene carrier for both in vitro and in vivo administration.⁹ However, the toxicity of PEI has been the chief drawback in its application.¹⁰ The enhancement of DNA condensation and DNA release has been achieved by combining two condensing agents while preparing the polyplex, which also provides lower cytotoxicity compared to when PEI is used as the sole condensing agent.¹¹

In particular, poly(ethylene glycol)-modified (PEGylated) PEI-based polyplexes are promising for in vivo gene therapy applications as potential non-viral vectors.¹² They are formed through a self-assembled electrostatic interaction between plasmid DNA (pEGFP) and PEG-b-polycation copolymers and are configured as polyplex micelles with a core–shell structure.¹³ The outer hydrophilic PEG segments enhance the micelle stability in serum, inhibits aggregation of polyplex, reduce protein opsonization of polyplex, improve the pharmacokinetic properties, and reduce the toxicity.^13,14 Recently, cyclic RGD peptides [c(RGDyK)] were incorporated onto the surface of the polyplex micelles for specific targeting to the tumor neo-vasculature.¹⁵ The RGD peptide (Arg-Gly-Asp) is a recognition motif in multiple ligands of α_v integrins and exhibits an increased affinity to α_vβ₃ and α_vβ₅ integrin receptors,¹⁶ which play a vital role in tumor angiogenesis and metastasis.^17–19 Enhanced cellular uptake and improved intracellular trafficking were achieved in polyplex micelles in the perinuclear region when RGD was conjugated to polyplex micelles, which improved transfection efficiency in HeLa cells possessing α_vβ₃ and α_vβ₅ integrins.²⁰ In particular, radiolabeled (^99mTc, ⁶⁸Ga, ¹⁸F, ⁶⁴Cu and ¹¹¹In) cyclic RGD peptides have been assessed over the last several years as targeted radiotracers for integrin α_vβ₃-positive tumor imaging by tomography techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET).²¹ Multimeric cyclic RGD moieties were exploited to increase the binding affinity of integrin α_vβ₃ to the radiotracer and were designed to be targeted radiotracers for imaging tumors.^22–24 Furthermore, the radiolabeled uptake (^99mTc, ⁶⁸Ga, ¹⁸F, ⁶⁴Cu and ¹¹¹In) of the multimeric cyclic RGD peptides is increased in various normal organs, such as the kidneys, liver, lungs and spleen, which may be due to increased peptide multiplicity.²⁵ Though it show an appreciably higher tumor uptake with a longer tumor retention time have been attained than their monomeric analogues, stills more efficient, stable and selective integrin α_vβ₃-targeted radiotracers are needed to enhance tumor uptake and increasing tumor-to-background ratio for early diagnosis of integrin α_vβ₃-positive tumors.

Here, we have demonstrated the preparation of targeting polymeric gene carriers based on dimeric cyclic RGD binding PEG-g-PEI, (E[c(RGDyK)]₂-PEG-g-PEI) based polyplexes, and evaluated their ^99mTc-labeled complex as a new radiotracer for in vivo studies. The PEG links were used to improve the radiotracer excretion kinetics from noncancerous organs, such as kidneys, liver and lungs. In particular, ^99mTc labeling is an essential focus due to its optimal nuclear characteristics such as half-life, low-cost abundance and γ-photon energy. The biophysical characterization of radiolabeled polyplexes was evaluated through various methods, including determination of the condensation efficiency, biodistribution, in vitro stability and in vivo application of the polymers. These properties were studied as function of time in 3D-SPECT/CT images and by end-point scintillation counting.

Materials and methods

Materials and reagents

Branched polyethylenimine [b-PEI, molecular weight 25 000 dalton] was purchased from Sigma-Aldrich. Methoxy poly(ethylene glycol)-N-hydroxysuccinimide [mPEG-NHS, molecular weight 2280 dalton] and N-hydroxylsuccinimide-PEG-maleimide [NHS-PEG-MAL, molecular weight 2100 dalton] were received from Creative PEG Works, Taiwan. Ethidium bromide (EtBr), agarose, uranyl acetate [UA], 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) and stannous tartrate were purchased from Sigma-Aldrich. ^99mTc pertechnetate was eluted from a technetium generator transferred from Global Medical Solutions Taiwan. Other common chemicals and solvents were purchased from Sigma-Aldrich and were used without further purification.

Synthesis of PEG-g-PEI

The preparation of the PEG-g-PEI copolymer was carried out in one step (Fig. S1†). The NHS group of monofunctional PEG reacted with the primary amine of PEI in H₂O under the N₂ atmosphere for 24 h. The degree of incorporation of PEG into PEI (i.e., PEGylation) was determined by ¹H-NMR from the ratio of the areas peaks corresponding to the –CH₂– protons of PEI (2.5–3.1 ppm) and PEG (3.3–3.7 ppm). Based on this estimate, copolymers have been prepared for different molar ratios of PEG : PEI of 1 : 1 (PP1), 3 : 1 (PP3), and 10 : 1 (PP10).

Dimeric cRGD peptide moieties were introduced to the PEG segment as the terminal group; the product is referred to as E[c(RGDyK)]₂-PEG-g-PEI (RPP). Another PEGylated polyethylenimine (PEI) copolymer was prepared without peptide moieties as end groups and is referred as PEG-g-PEI (PP).

Synthesis of E[c(RGDyK)]₂-PEG-g-PEI

The synthesis of E[c(RGDyK)]₂-PEG-g-PEI (RPP) is a three-step reaction (Fig. S2†). In the first step, the amino group of the lysine residue of E[c(RGDyK)]₂ was altered to a thioacetyl group via reaction with Traut's reagent in HEPES buffer (20 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 7.2) with various molar ratios from 1 to 5. The reaction mixture was incubated under argon at room temperature for 2 h. In the second step, NHS-PEG-maleimide was subsequently mixed with a certain amount of PEI under argon for 4 h in HEPES buffer. The thioacetyl group of the E[c(RGDyK)]₂ was reacted with the maleimide group of the PEG-g-PEI under argon overnight. Both the PP and RPP products were purified by centrifugal filtration (10 000 MWCO, Millipore), washed with H₂O for 15 min and lyophilized to a dry powder.

Formation of polyplex

Polyplexes were prepared with PEG-g-PEI and plasmid EGFP (pEGFP) at different N/P (w/w) ratios, such as 0.1, 0.2, 0.5, 1, 2 and 5. The N/P ratio is the polyplex composition measured as the total weight of the polycation to the weight of pEGFP. After gently vortexing the sample, PEG-g-PEI/pEGFP (PP/D) polyplexes of a uniform size were formed and then incubated for 30 min.

Characterization of copolymer

The composition of the prepared PEG-g-PEI copolymers was estimated using ¹H NMR (Bruker AM-500 NMR). The particle sizes and the PEI/D, PP/D and RPP/D zeta potentials were measured using dynamic light scattering (DLS, Zetasizer 3000HS). The morphology of PEG-g-PEI/DNA polyplex gene delivery vehicles was observed using a Hitachi H-600 transmission electron microscope. To understand the interaction ability between the positively charged b-PEI and the negatively charged pEGFP with different N/P ratios, a gel retardation assay using electrophoresis was performed. Aliquots of the solution were taken and assayed by electrophoresis through a 0.7% agarose gel at 80 V for 40 minutes, and the DNA was visualized on a UV transilluminator (or UV box) after immersing the gel in ethidium bromide (EtBr) solution. The cell viability assay studies were evaluated according to a previously reported method.²⁶ Flow cytometry was performed as described by Ogris et al.²⁷ All animal experiments were performed in compliance with the guidelines of the Animal Committee of the National Tsing Hua University and all experimental protocols were approved by the Animal Committee of the National Tsing Hua University but human consent had not been used.

In vitro transfection

100 000 cells per well were seeded in the 6-well plates which would reach 70–80% confluence onto transfection one day later. A certain amount of PEI, PP and RPP were added to the PBS solution with a fixed amount of plasmid DNA, followed by gentle vortexing for 30 seconds. The resulting solution was incubated at room temperature for 30 minutes and then diluted with growth medium to different concentrations. Before transfection, the old medium was removed from the 6-well plates, followed by washing with PBS three times. Then the polyplex was added and incubated at 37 °C under 5% CO₂ atmosphere for 24 h or 48 h. The fresh growth medium containing 10% FBS was added. The cells were incubated for 24 h in an incubator at 37 °C in 5% CO₂. Then the cells were washed once with PBS and then detached with 0.25% trypsin/PBS. Transfection efficiency was evaluated by measuring the percentage of cells expressing GFP (represent G_mean value) using a FACSCanto™ System (Becton-Dickinson, San Jose, CA). Fluorescence parameters from 10 000 cells were acquired. The transfection experiments were carried out in triplicate. The GFP expression was directly visualized under the fluorescence microscope after autofluorescence subtracted.

PP/D and RPP/D polyplex binding assay by flow cytometry

To understand the binding abilities of the PP/D and RPP/D polyplexes, the fluorescent polyplexes were used to detect the binding signals with a FACSCanto™ System by using the signal of the cyanine dyes 7 (Cy7) with ex/em: 743 nm/767 nm as the binding signal, while the GFP signals, ex/em: 395 nm, 475 nm/509 nm, were utilized for the reporter gene expression. Cell binding and uptake efficiency were evaluated by measuring the percentage of cells expressing Cy7 using a standard filter of Alexa Fluor® 700 in the FACSCanto™ System. Fluorescence parameters from 10 000 cells were acquired, and the studies were carried out in triplicate.

Radiolabeling polyplexes with ^99mTc

To a lyophilized vial containing strong ligand (i.e., PEI), 0.1 mg of stannous tartrate in 1 ml of H₂O was added and mixed under N₂. Immediately, a [^99mTc] sodium pertechnetate solution (40 mCi) in 0.9% saline was added. After adding ^99mTcO⁴⁻, Tc⁷⁺ is reduced by Sn²⁺ ions, and the reduced ^99mTc readily forms a chelate with the weaker ligand, i.e., ^99mTc-tartrate. Then, the stronger ligand (PEI) readily reacts with ^99mTc-tartrate by ligand exchange to form ^99mTc-polyplex. Radiochemical purity and radiolabeling efficiency was measured using silica-gel impregnated glass fiber instant thin-layer chromatography (ITLC-SG) by using acetone as the mobile phase, and the radioactivity was counted using an automatic gamma counter (1470 WIZARD). The PEG-g-PEI/pEGFP-^99mTc or E[c(RGDyK)]₂-PEG-g-PEI/pEGFP-^99mTc polyplexes (PP/D-^99mTc or RPP/D-^99mTc), which have large molecular weights, would remain at the origin while the pertechnetate moves with the mobile phase at the solvent front.

Radiolabeling stability

The stability of the ^99mTc labeled polyplexes in physiological saline and rat plasma was determined at different time intervals using ITLC-SG. The ^99mTc labeled polyplexes were mixed in saline for 30 minutes, 0.1 ml of the solution (about 0.5–1 mCi) was withdrawn and 0.9 ml of saline or plasma was added in a 1 ml vial. The solution mixture was incubated in saline at room temperature for a period of time from 3, 6, to 24 h. On the other hand, the same solution were prepared and incubated in rat plasma at 37 °C for a period for time from 3, 6, to 24 h. After being incubated for different times, the ^99mTc labeled polyplexes solutions in saline or plasma were checked for radiochemical activities using an automatic gamma counter.

NanoSPECT/CT imaging

A high-resolution dual-modality system NanoSPECT/CT (Bioscan, Inc., Washington, DC) imaging, equipped at Chang-Gung Memorial Hospital, Linkou, Taiwan, was used for the animal imaging study. This system features helical scanning for both single-photon emission computed tomography (SPECT) and CT acquisitions using a translation stage with variable axial scanning range from 30 to 180 mm. The static SPECT/CT imaging was taken for whole body scan with 24 projections and every projection acquired signal within 60 seconds. A total of three mice were respectively injected intravenously into the tail vein each with 1–2 mCi/0.2 ml and 40 μg of ^99mTc-labeled polyplex. The mice were anesthetized with 1.5% isoflurane, positioned prone in the scanner, and performed with nanoSPECT/CT imaging at 1, 3, 6, and 24 h after intravenous injection via the tail vein. All SPECT images were reconstructed using the iteratively ordered subset expectation maximization method from overlapping projects.

Image analysis

Images were viewed and quantified using PMOD image analysis workstation (PMOD Technologies Ltd., Zurich, Switzerland): three-dimensional regions of interest (ROI) on the CT images were drawn around the liver, kidney, lung, heart, spleen, bone, and tumor on decay-corrected whole-body coronal images. The average radioactivity concentration (accumulation) within a tumor or an organ was obtained from mean pixel values within the ROI volume, which were converted to counts per millimeter per minute by using a conversion factor. Assuming a tissue density of 1 g ml⁻¹, the counts per gram per minute and then divided by the injected dose (ID) to obtain an imaging ROI-derived percent injected dose per gram of organ or tissue (% ID/g or % ID/cm³).

Results and discussion

Syntheses and characterizations of PEG-g-PEI and E[c(RGDyK)]₂-PEG-g-PEI copolymers

RGD-peptides are good targeting ligands for the tumor endothelium using polyplexes containing siRNA and pDNA. Assorted pathogenic organisms were used for an integrin targeting approach by using an integrin receptor to promote their cellular internalization.^18,28 Although RGD-PEG-PEI has been observed to efficiently deliver nucleic acids into tumors, it has not demonstrated the enhanced uptake with modulated intracellular trafficking that was observed with cRGD.²⁹ Thus far, RGD-mimetics have been used as therapeutic agents such as angiogenesis inhibitors, imaging tracers for atherosclerotic plaques, inhibitors for macrophages and enhancers of osseointegration.^30,31 Here, we describe the synthesis of an RGD-conjugated PEG-g-PEI copolymer with different molecular compositions.

The formation of the PEG-g-PEI copolymer with different ratios of PEG to PEI was confirmed and the compositions of each fraction were determined using ¹H-NMR; the results are shown in Fig. 1. The peak at approximately 2.5–3.1 ppm corresponds to PEI hydrogens and the peaks at approximately 3.3–3.7 ppm are attributed to PEG and reveal the existence of both PEG and PEI in the copolymer. The molar ratios are calculated by counting the number of hydrogens both on PEI at 2.5–3.1 ppm and on PEG at 3.3–3.7 ppm. Accordingly, three different copolymers with PEG/PEI ratios close to 1 : 1, 3 : 1, and 10 : 1 are shown in Table 1. The cRGD conjugated PEG-g-PEI copolymer was synthesized, consisting of a 25 kDa molecular weight PEI as the core and a heterobifunctional PEG linker for the PEGylation. PEG was decorated with cRGD molecules at the distal end of the PEG chains, as shown in Fig. 1b. Coupling of NHS-PEG maleimide to PEI in HEPES buffer was confirmed by ¹H NMR, and the degree of coupling was determined to be approximately 99% of the feed. Hence, conjugation of cRGD to activated PEG depended on both an excess of cRGD moieties and the amount of PEG-linker present. To prepare cRGD-conjugated PP, a 5-fold molar excess of PP and a 4-fold molar excess of RGD were used, resulting in a conjugate designated as E[c(RGDyK)]₂-PEG-g-PEI (RPP). Similarly, E[c(RGDyK)]₂-PEG-g-PEI copolymers were identified by the presence of a peak at 7 ppm due to the tyrosine hydrogen of the E[c(RGDyK)]₂ moiety. The molar ratio of PEG to E[c(RGDyK)]₂ was calculated to be 19.6 from the integrated number of hydrogens at 3.3–3.7 ppm and at 7 ppm in the ¹H-NMR spectra.

Fig. 1 ¹H NMR spectra of the synthesized copolymers and RGD-bound copolymer in D₂O: (a) PEG-g-PEI and (b) E[c(RGDyK)]₂-PEG-g-PEI.

Table 1 The molar ratios of PEG/PEI and PEG/RGD of PEG-g-PEI copolymers from the reactants to the products

Copolymer	Reaction		Production
	PEG/PEI	PEG/RGD	PEG/PEI	PEG/RGD
	(mol/mol)	(mol/mol)	(mol/mol)	(mol/mol)
PEG-g-PEI (PP1)	1	—	1.1	—
PEG-g-PEI (PP3)	3	—	3.6	—
PEG-g-PEI (PP10)	10	—	9.9	—
E[c(RGDyK)]2-PEG-g-PEI (RPP10)	10	8	10.8	19.6

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Formation of PEG-g-PEI/DNA polyplexes

PEGylated PEIs and cRGD-modified PEI copolymers are known to be capable of forming polyplexes with pDNA. Obviously, the polyplex complex formation can be influenced by the spatial orientation of the targeting moiety. This orientation may be toward the surface of the complexes, resulting in the formation of spherical particles. Polyplex self-assembled micelles were prepared by mixing each polymer solution with the pDNA solution at various N/P ratios. The N/P ratio was calculated based on the number of amino groups on the polymer participates in DNA condensation. To demonstrate the interaction ability between positively charged b-PEI and negatively charged pEGFP with different N/P ratios, a gel retardation assay was performed using 0.7% agarose gel. The movement of the plasmid in the gel would be slowed as the amount of the PEG-g-PEI increased, owing to the neutralization of negative charges on the DNA bound in the polymer. At N/P ratios exceeding the ratio for sufficient neutralization, the formed polyplex would migrate slightly toward the anode, providing evidence that it is positively charged. Polyplexes were prepared with PP and pEGFP at different N/P ratios: 0.1, 0.2, 0.5, 1, 2 and 5. EtBr have been used to produces a strong fluorescence signal when intercalating with DNA. When DNA is condensed by conjugation with a polycation, ethidium bromide cannot intercalate with DNA. Upon the pDNA condensation, decreased fluorescence emission has been obtained due to the coil-to-globular structural transition, and the globular structure inhibits the intercalation of DNA by EtBr. Hence, EtBr exclusion assay have been used to determine the condensation degree of pDNA at different N/P ratios quantitatively. As shown in Fig. 2, the band of free pDNA disappeared at N/P ratio of 0.5 in all of the samples, indicating successful and complete polyplex formation between pDNA and the polycation. Additionally, PEG side chains had no significant effect on the polyplex formation between the PEI main chain and the anionic pDNA.

Fig. 2 Agarose gel electrophoresis of PEI/DNA and copolymer/DNA complexes at various N/P ratios. Lane 1, DNA marker [excluded (a)]; lane 2, N/P = 0 (plasmid only); lanes 3–8, N/P ratio: 0.1, 0.2, 0.5, 1, 2 and 5.

Polyplexes are aggregates and have to be evaluated in the context of surface charge. Therefore, zeta potential measurements can provide sufficient information about polyplex formation by the plasmid and the PP, owing to the neutralization of negative charges on the DNA bound to the positively charged polymer backbone. The zeta potentials of PP/DNA polyplexes at different N/P ratios were measured in H₂O and are shown in Fig. 3. The zeta potentials of the polyplexes increased with increasing N/P ratios but showed slight difference at high weight–weight ratio. A more positive surface charge is observed in PP polyplexes with higher N/P ratios due to the shielding effect by nonionic PEG.³² Most likely, PEG moieties react with the 2° amines of PEI, while copolymerization with 3° amines is hindered due to their high electron density and lower steric hindrance. With PEGylation, the chance to protonate is lower than that observed for 2° amines due to the conversion from 2° amine to 3° amines. Thus, variation in the surface charge of polyplexes may be controlled by decreasing the degree of protonation.

Fig. 3 Zeta potential measured for the prepared polyplexes with DNA (PEI/D, PP1/D, PP3/D and PP10/D).

The polyplexes synthesized with N/P ratios of 0.1 and 0.2 exhibited net negative charges because the concentration was insufficient to form a complex and condense DNA. While at 0.5 N/P w/w ratios all polyplex shows positive zeta potential, which is revealed that all DNA are condensed in polycation of PEI. This finding also supported with the result obtained from electrophoresis analysis and confirmation for successful and complete polyplex formation at 0.5 N/P weight ratios as shown in Fig. 2. Thus, for the formation of polyplexes with an integral morphology and a lower positive charge have been obtained when N/P ratio = 1, which was selected for generating the efficient PP/DNA polyplex for further analysis.

Cell viability assay

High-molecular-weight PEI (approximately 25 kDa) is significantly more toxic than low-molecular-weight PEI;³³ however, high-molecular-weight PEI has a higher transfection efficiency than low-molecular-weight PEI. To decrease the toxicity of high-molecular-weight PEI, the introduction of PEG to modify PEI is an effective method. Thus, PEGylation is known to help reduce the cytotoxicity of PEI by decreasing the number of PEI amino groups.³⁴ Moreover, PEI was proven to effectively condense pEGFP, while the formation of the polyplex and the stability of the complex depended on the degree of PEG grafting.³⁵ To investigate the cytotoxicity of the PEI and PP copolymers, cell viability was determined at different concentrations in HeLa cells. Free polymers were used to measure the cytotoxicity rather than DNA complexes owing to the reduced toxicity that may be obtained by the formation of DNA polyplexes.³⁶ The cytotoxicity of PEI and PP copolymers at different concentrations in HeLa cells were evaluated (Fig. 4). It was found that the cell viability decreased drastically when the concentration of 25 kDa PEI, and PP copolymer was increased from 0.01 to 0.05 mg ml⁻¹, whereas approximately >85% of the cell viability could be maintained at a low concentration of PEI and PP copolymer, i.e., less than 0.01 mg ml⁻¹. At a high concentration (0.05 mg ml⁻¹) of either PP1/D or PP3/D, the cell viability decreased to about 70%, whereas PEI/D shows <40% cell viability. At all concentration (0.01–0.05 mg ml⁻¹), PP10/D shows high cell viability, i.e., above 89%. As shown in Fig. 4, cell viability was follows the orders as PP10/D > PP3/D > PP1/D > PEI/D, which is revealed that the toxicity of PEI was apparently correlated with the degree of PEG grafting and that PP10/D complexes show high cell viability.

Fig. 4 Cytotoxicities of PEI/DNA and copolymer/DNA polyplexes at varying concentrations of 0.001, 0.005, 0.01, 0.025, and 0.05 mg ml⁻¹ (n = 4).

Transfection efficiency of the PEG-g-PEI/DNA polyplexes

The transfection efficiency from flow cytometry was determined by detecting the fluorescence intensities (G_mean) of the cells. As seen from Fig. 5, PP10/D showed the highest transfection efficiency compared to the other two polyplexes with a concentration of 0.025 mg ml⁻¹. It was a surprise that PEI/D at 0.025 mg ml⁻¹ showed much lower transfection efficiency, although it had a high transfection capability due to its inherent polycation nature. This polycation polymer had aroused the death of the cells suspended in the medium, apparently due to its high toxicity. The dead cells were excluded from the flow cytometry count because of its nonspecifically bind with many reagents, which can lead to false positive results. In addition, dead cells may also uptake fluorescent probes since their membranes are permeable to reagents and generate artifacts results.

Fig. 5 Transfection efficiencies of the polyplexes with different PEG/PEI ratios at varying concentrations of either copolymer or DNA (N/P = 1).

The toxicity of PEI would reduce the transfection efficiency, as evidenced by the fact that a lower concentration (0.01 mg ml⁻¹) resulted in a higher transfection efficiency compared with the concentration of 0.025 mg ml⁻¹. The transfection efficiency was clearly affected by the PEG/PEI ratios of the polyplexes; the higher ratio case, i.e., PP10/D, showed much higher efficiency (about 68%), indicating the PEG to PEI sheltering effect.²⁶ As expected, it was demonstrated that higher concentrations of the polyplex correlated with higher transfection efficiency. Thus, PEI, PP1 and PP2 complexes exhibited lower transfection efficiency compared to PP10 due to sheltering effect from sufficient PEG segments to PEI segments. At low concentration (0.005–0.001 mg ml⁻¹), transfection efficiency of all polyplex was decreased, which may due to insufficient condensation and protection of DNA inside the complex.

Morphology and size of the PEG-g-PEI/DNA polyplexes

The size and shape of a polyplex is acknowledged to be one of the major factors in transfection efficiency. As shown in Fig. 6, the size of the self-assembled PP/DNA polyplex is compared with the polymers by measuring their hydrodynamic diameters. The particle sizes of both the PEI- and PP-based polyplex are quite similar, and the copolymerization of PEI does not affect their self-assembled properties with the addition of pDNA. Thus, the particle sizes of the polyplexes with different PEG/PEI ratios but the same N/P ratio of 1 were apparently similar over the range of 90 to 140 nm, which may reveal that the N/P ratio and the PEG/PEI ratio would not be the key factors for determining the particle size of the synthesized polyplexes. Interestingly, the cRGD-modified PP-based polyplex (RPP10/D) seemed to have a larger particle size of approximately 135 nm due to the larger size of the cyclic RGD, which may influenced by the protonation profile of the PEI segments, the protonation of the targeting ligand and their interaction with polyanions. Moreover, coupling of PEG decreased the electrostatic interaction of PEI with the negatively charged DNA, which seemed to decrease the size of the polyplexes. The morphology of PP/DNA polyplexes was spherical, as evidenced by the TEM results shown in Fig. 6b.

Fig. 6 (a) Particle sizes of PEI/D and copolymer/DNA polyplexes at N/P = 1 (n = 3). (b) A representative image for confirmation of the PP10/DNA polyplexes imaged by TEM.

Cell binding assay

Because previous studies showed that the PP10 copolymer had high transfection efficiency and high viability, this copolymer has been conjugated with E[c(RGDyK)]₂ to render it a targeting gene delivery carrier; the conjugated product is referred to as RPP10. To confirm the specific cell binding and transfection efficiency of RPP10, a parallel assay for cell binding using PP10 and RPP10 was carried out with flow cytometry, and the results are presented in Fig. 7. PP10/D and RPP10/D were prepared at a concentration of 0.025 mg ml⁻¹ of either copolymer or DNA (N/P = 1) and labeled with the fluorescein Cy7. After incubation of PP10/D-Cy7 or RPP10/D-Cy7 with HeLa cells for 1, 3, and 18 h, the binding of PP10/D-Cy7 and RPP10/D-Cy7 in the cells was analyzed via cell binding signals; the binding of RPP10/D-Cy7 was significantly higher than that of PP10/D-Cy7 (Fig. 8a). As presented in Fig. 8a, the left black peak was from a control maintained at a low fluorescence intensity, the middle green peak was from the cells accumulated with PP10/D-Cy7, and the right red peak was from the cells accumulated with RPP10/D-Cy7. The results indicated that the gene vector with the E[c(RGDyK)]₂ targeting ligand could enhance the binding ability of the HeLa cells. To compare the binding ability and transfection efficiency of RPP10/D with PP10/D, the HeLa cells were subsequently incubated with PP10/D-Cy7 and RPP10/D-Cy7 for 18 h, and GFP expression signals were detected by flow cytometry. It was seen from the results Fig. 8b that the binding ability of the cells with RPP10/D was higher than that of the cells with PP10/D. Polyplexes are exposed to high local concentrations of charged macromolecules when they interact with cells, demonstrating that competitive binding with polycations and polyanions within the cell can weaken the polyplexes and cause nucleic acid to be released within the cell. For that reason, GFP expression signals of gene vectors with the E[c(RGDyK)]₂-conjugated RPP targeting ligand could enhance the transfection efficiency. All of these results demonstrated that RPP10/D can transport DNA into the cells effectively and might be utilized as an efficient gene delivery vector.

Fig. 7 Cell binding and uptake abilities of PP10/D-Cy7 and RPP10/D-Cy7 by the HeLa cells along with incubation for 1, 3, and 18 h (N/P = 1, in 0.025 mg ml⁻¹, n = 3). Error bars represent SD. *P < 0.05; the results for G_mean of RPP10/D are significantly higher than those of PP10/D.

Fig. 8 (a) Flow cytometry chromatograms of the HeLa cells incubated with RPP10/D-Cy7 and PP10/D-Cy7 for 18 h. (b) Comparison of the transfection efficiency of RPP10/D with PP10/D from the incubation of the cells with both of the gene vectors for 18 h and G_mean for RPP10/D was higher than for PP10/D (*P < 0.05).

Radiolabeling of PP10/D and RPP10/D by ^99mTc

Radiochemical purity and radiolabeling efficiency was measured using silica-gel impregnated glass fiber instant thin-layer chromatography (ITLC-SG). Radiolabeling efficiencies of PP10/D and RPP10/D with ^99mTc was analyzed and shown to be 96.87% and 98.54%, respectively. The radiochemical stabilities of PP10/D-^99mTc and RPP10/D-^99mTc during incubation in normal saline at room temperature and in rat plasma at 37 °C are summarized in Table 2. PP10/D-^99mTc and RPP10/D-^99mTc in either condition retained more than 90% radiochemical purity after incubation for 6 h and more than 80% radiochemical purity after incubation for 24 h. It was confirmed that the facile labeling method could produce PP10/D-^99mTc and RPP10/D-^99mTc with high radiolabeling efficiency and high stability in either of PBS or plasma.

Table 2 Stabilities (%) of PP10/D-^99mTc and RPP10/D-^99mTc during incubation in normal saline and rat plasma for 3, 6, and 24 h

	Incubation time (h)	3	6	24
PP10/D-^99mTc	In saline	95.93 ± 2.81	95.64 ± 1.72	87.97 ± 5.26
	In plasma	92.14 ± 5.02	90.45 ± 8.92	80.88 ± 6.92
RPP10/D-^99mTc	In saline	97.29 ± 0.16	96.75 ± 0.29	85.66 ± 0.48
	In plasma	96.12 ± 0.98	95.52 ± 2.75	84.21 ± 9.80

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Body distribution

RPP10/D polyplexes exhibited significantly higher binding affinity and transfection efficiency (Fig. 8) due to effective DNA transportation into the cells. Thus, RPP10/D polyplexes can be identified as promising candidates for local in vivo gene transfer due to their improved transfection efficiency. There was a time-dependent change in the distribution of intravenously injected ^99mTc-labeled RPP10/D polyplex complexes in the heart, lungs, liver, kidney, spleen, intestine, blood and brain of female BALB/c nude mice bearing subcutaneous HeLa tumors, as measured through coronal nanoSPECT/CT imaging (Fig. 9). The images from left to right are CT, nanoSPECT and nanoSPECT/CT. The tumors were clearly visualized with good contrast in both animal models, and the positions of HeLa tumors are indicated by white arrows. We found that the two tracers of radioactive complexes mainly accumulated in the liver, spleen and kidneys at 1, 3, 6, and 24 h after intravenous injection. Uptake in the spleen and the liver is a common feature of the distribution of the nanoparticles in mice, as studied previously,³⁷ due to large concentration of macrophages which are parts of the reticuloendothelial systems (RES).

Fig. 9 Comparison of coronal nanoSPECT/CT images of PP10/D-^99mTc and RPP10/D-^99mTc in female BALB/c nude mice bearing subcutaneous HeLa tumors. The images were acquired at 1, 3, 6 and 24 h after intravenous injection. PP10/D-^99mTc or RPP10/D-^99mTc containing approximately 1.2 mCi of ^99mTc was administered to the mice by intravenous injection. The images from left to right are CT images, nanoSPECT images and nanoSPECT/CT images. The positions of HeLa tumors are indicated by white arrows.

The coronal nanoSPECT/CT images of the two tracers showed the accumulations in the tumor at 1 h after intravenous injection. The coronal nanoSPECT/CT images of RPP10/D-^99mTc showed accumulations in the tumor until 24 h. In contrast, the coronal nanoSPECT/CT image of PP10/D-^99mTc showed almost no accumulation in the tumor at 24 h post-injection. To reduce the sacrifices of animals for in vivo studies, SPECT/CT imaging was utilized for the biodistribution study. Regions of interest (ROI) of organs or tissues were circumscribed, and the percentages of doses were calculated. The data from the quantitative analysis of the tracer uptakes in the tumors and the other organs or tissues from SPECT imaging are shown in Fig. 10. The tumor uptakes observed on the basis of SPECT quantification are 31.1, 19.8 and 14.6% ID/cm³ at 1 h for PP10/D-^99mTc in female BALB/c nude mice bearing subcutaneous HeLa tumors in the liver, kidney and spleen tumor, respectively. The tumor uptake value of RPP10/D-^99mTc was higher than that of PP10/D-^99mTc at 1 h after injection and at the subsequent time points post-injection. The accumulation of the two tracers in the lungs was not higher than the accumulations in the heart, liver, kidney and spleen. This means that PP10/D or RPP10/D with PEI being well sheltered by PEG could invade from the lung uptake.³⁸ We also noticed the fact that the accumulation of the targeting tracer in bone tissues was higher than the accumulations in the heart, lung, liver and spleen at 3, 6 and 24 h. This is in agreement with a previous report.³⁹ The bone uptake may be due to the expression of α_vβ₃ integrin in the bone, which is targeted by the tracer. In general, the bone imaging agents (e.g., ^99mTc–phosphonate complexes) depend on ion exchange to accumulate in the bone.⁴⁰ The gene vector imaging was also observed to be effectively accumulated in the bone, being evidenced as the targeting effect of the E[c(RGDyK)]₂-bound copolymer.

Fig. 10 (a) In vivo biodistribution data of PP10/D-^99mTc in female BALB/c nude mice bearing subcutaneous HeLa tumors. The values are presented as % ID/cm³, mean ± S.D. (b) In vivo biodistribution data of RPP10/D-^99mTc in female BALB/c nude mice bearing subcutaneous HeLa tumors. The values are presented as % ID/cm³, mean ± S.D. (n = 3 at each time point).

The main excretion routes for PP10/D-^99mTc were both urinary and hepatobiliary, but RPP10/D-^99mTc seemed to be mainly excreted via the urinary system. The liver and spleen uptakes for RPP10/D-^99mTc were apparently lower than those for PP10/D-^99mTc. As a result, the average tumor-to-liver ratio was higher for RPP10/D-^99mTc than for PP10/D-^99mTc at every post-injection time point. The average tumor-to-kidney ratio was slightly higher for RPP10/D-^99mTc than for PP10/D-^99mTc at every post-injection time point. The tumor-to-heart (tumor-to-blood pool) ratios for RPP10/D-^99mTc were clearly higher than those for PP10/D-^99mTc at the same post-injection time points, with the highest ratio at 24 h post-injection (Fig. S3 and S4†). It is revealed from the results that the targeting ligand, E[c(RGDyK)]₂, can improve the tumor accumulation and accelerate the clearance of the bound polyplex from the main organs or tissues. Overall, our results demonstrated that the PP10/D-^99mTc and RPP10/D-^99mTc biodistribution trends studied with the targeting ligand E[c(RGDyK)]₂ could improve the tumor accumulation, providing evidence of its targeting effect. Hence, RPP10/D can transport DNA into the cells effectively and might be utilized as an efficient gene delivery vector.

Conclusion

The findings of the present study demonstrate the feasibility of using a dimeric cyclic RGD-PEG-g-PEI/pEGFP polyplex labeled with ^99mTc as a radiotracer for tumor imaging. The polyplex with a high PEG/PEI ratio of 10 : 1 can condense with DNA effectively as a gene delivery vehicle. PEG-g-PEI copolymer, upon conjugation with DNA with a ratio of 1/1 (w/w), was suitable for DNA condensation, as evidenced by agarose gel retardation assay and size and zeta potential measurements. The RPP10/D polyplex exhibited significantly higher binding with the HeLa cells and enhanced gene expression compared with non-targeting PP10/D. The RPP10/D polyplex demonstrated significantly higher binding affinity and transfection efficiency and exhibited effective endosomal escape properties due to protonation of branched PEI chains with decreasing pH. Polyplexes as molecular imaging probes are observed using SPECT/CT imaging; the uptake of RPP10/D in the tumor was higher than that of PP10/D. The obtained results revealed that the interaction of the ^99mTc-labeled targeting ligand E[c(RGDyK)]₂-conjugated PEG-g-PEI with tumor tissue resulted in a strong signal, which means that the tumor uptake of radiotracers in the in vivo PET images and biodistribution of the tumor.

Acknowledgements

We are indebted to Ministry of Science and Technology, Taiwan, for financial support (MOST 103-2221-E-033-012-), and thanks Jem-Mau Lo and Jian-Yu Chen for his kindly assistance in this study.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18644g

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