PEG-Based Hyperbranched Polymer Theranostics: Optimizing

Jul 25, 2014 - MTS-Capped HPB. End-group aminolysis was performed in the presence of methylmethanethiosulfonate (MMTS) using the following procedure: ...
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PEG-Based Hyperbranched Polymer Theranostics: Optimizing Chemistries for Improved Bioconjugation Aditya Ardana,† Andrew K Whittaker,†,‡ and Kristofer J. Thurecht*,†,‡ †

Australian Institute for Bioengineering and Nanotechnology and Centre for Advanced Imaging and ‡ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Queensland, St. Lucia, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Polymeric materials are receiving increasing attention as gene delivery vectors in nanomedicine. Because of their potential to combine many different functionalities into a single molecule, hyperbranched polymers (HBPs) are ideal for biomedical applications and have been utilized in a variety of applications ranging from molecular imaging to vectors for gene and drug delivery. In this study, we synthesized a model functional HBP as a potential theranostic, which can act as an imaging agent for 19F MRI while at the same time carrying specific therapeutic genes (such as smallinterfering RNA) to a site of interest. In order to realize such a goal, an in-depth analysis of the molecular structure of the theranostic was performed to ensure that the diagnostic (19F MRI) and therapeutic (gene therapy) components were complementary and did not compromise respective individual function. Importantly, it was necessary to demonstrate that the various chemistries utilized during the synthesis of the theranostic HBPs were compatible and did not lead to unwanted degradation and subsequent formation of side-products. Ultimately, we show that through careful analysis of the polymeric materials we can gain an understanding of the subtle factors that influence successful development of a theranostic device that incorporates multiple specific functionalities.

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INTRODUCTION Polymeric devices have evolved as extremely promising means of promoting therapeutic benefit.1,2 However, truly effective polymeric devices must incorporate new therapies, imaging, and targeting moieties, while maintaining their physical and chemical integrity throughout the synthetic procedure. Central to the development of these future therapeutic platforms is the field of theranostics. Theranostics promise the ability to deliver a therapeutic dose to the correct site within the body, but they also possess mechanisms for real-time diagnosis, monitoring of disease progression and visualization of drug delivery, release and efficacy of treatment.3−5 The field of theranostics has advanced rapidly over the previous decade and this has been fueled both by significant improvements in imaging technology and by the availability of far more sophisticated techniques for synthesizing complex and multitasking delivery vehicles.6,7 Hyperbranched polymers (HBPs) are one class of dendritic polymer that exhibits high end-group functionality while maintaining good solubility.8 Although structurally similar to dendrimers, HBPs have the important advantage that they can be prepared in readily scalable quantities with highly diverse and variable functionalities.9 While various polymerization techniques can be applied to synthesize HBPs, much attention recently has been focused on utilizing reversible-addition−fragmentation chain transfer (RAFT) polymerization9,10 as a means of controlling the structural properties of the HBPs. This is mainly due to the implementation in RAFT polymerizations of mild reaction conditions, applicability to a wide range of monomers © 2014 American Chemical Society

and the ability to develop materials with well-defined end-groups that can be subsequently modified for biomedical applications.1,11−13 The potential to combine several different functionalities into a single molecule makes RAFT-synthesized HBPs ideal for biomedical applications. Indeed, hyperbranched polymers have been utilized in molecular imaging14−16 and also as vectors for gene and drug delivery.2,17 Recently, our group has developed HBPs as unique scaffolds for 19F MR imaging that show promise in vivo. However, despite their facile synthesis and attractive use in biomedicine, the complex structure of HBPs often becomes a limitation for their future application, especially in the nanomedicine field where it is essential that material properties are absolutely determined and controlled. For example, in order to achieve high signal-to-noise ratio in images, 19F MR agents require fluorinated segments to possess high mobility in physiological media. To achieve this, our group has typically developed materials in which the 19F-containing monomers are dispersed throughout a hydrophilic matrix (e.g., PEG-based monomers).15,16 While this leads to improved imaging performance, optimization of the imaging parameters can only be achieved if a true understanding of the sequence and end-group structure within the macromolecules is achieved, since these details will ultimately affect the molecular mobility, and hence Received: June 9, 2014 Revised: July 16, 2014 Published: July 25, 2014 5211

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Scheme 1. Synthetic Scheme of the Theranostic PEG-Based HBP

of large biomolecules (siRNA) to dense chain-ends (such as those in hyperbranched polymeric materials) was investigated and we report on methodologies for increasing the yield of these hybrid materials. The general synthetic approach is described in Scheme 1, where the synthesis of the HBP is described, followed by successive chemistries allowing modification of the chain-ends for attachment of siRNA.

imaging performance. The ability to fully characterize HBPs is crucial if an understanding of the link between structure and property is to be gained, and if this relationship is to be ultimately tuned through synthetic strategies.18 Gene therapy has emerged as an important method of treatment for many disease states.19,20 Control of gene expression by RNA interference (RNAi) is a means to downregulate a vast array of disease processes via a specific interference pathway. Research into RNAi for therapy (especially cancer related therapies) has advanced significantly, with the first reports of in vivo efficacy in humans published for siRNA in 2010.21 Nonetheless, in order for gene therapy to be a viable and universal therapeutic option, a number of important issues must be first addressed.22,23 Of particular importance is the poor in vivo stability of small RNAs due to the presence of degrading enzymes (RNase and DNase) within the blood plasma. Additionally, efficacy of cell transfection is often compromised due to poorly designed carrier vehicles (or in the case of free small RNAs, electrostatic repulsion from the cell membrane).24 In an attempt to overcome some of these problems, novel and effective approaches to delivery of small RNAs for gene therapy have evolved. Typically, this involves covalent attachment of the small RNA to a nonviral vector using a stimuli-responsive linker. In this way, the therapeutic payload can be released under specific intracellular stimuli.25−28 However, efficacious translation of this approach to animal models requires a more detailed understanding of factors that affect the number and density of RNA that is ligated to the polymers, in addition to studies that investigate the stability of the conjugates in vivo. In this study, we synthesized a functional HBP as a model for a potential theranostic containing an imaging modality (19F MRI) as well as the ability to act as a carrier for therapeutic genes. The aim of this research is to investigate whether the various chemistries that are utilized in the synthetic approach are complementary, particularly in regards to stability of the different modalities. In addition, factors surrounding the ligation efficiency

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EXPERIMENTAL SECTION

Materials. Poly(ethylene glycol) methyl ether methacrylate (PEGMA) (∼475 Da), ethylene glycol dimethacrylate (EGDMA), and trifluoroethyl acrylate (TFEA) were passed through alumina prior to use to remove inhibitors. 2-dodecylsulfanyl thiocarbonyl sulfanyl-2methyl propionic acid (CTA) and the alkyne analogue (CTA-alkyne) were synthesized as previously reported.29 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol three times before use. Tetramethylethylenediamine (TEMED) for gel electrophoresis was purchased from Bio-Rad Laboratories. Thiol-protected sense strand (5′-/5ThioMC6-D/rCrUrG rGrArC rUrUrC rCrArG rArArG rArArC rATT-3′) and the antisense strand (5′-rUrGrU rUrCrU rUrCrU rGrGrA rArGrU rCrCrA rGAA-3′) were purchased from Integrated DNA Technologies. Deprotection of the thiol group on the sense strand (100 μM) was performed in buffer KHCO3 (purchased from SigmaAldrich) at pH 9 in the presence of 100 μM dithiothreitol (DTT, purchased from Sigma-Aldrich) as outlined in the manufacturer’s protocols. DTT was removed from the thiol sense strand by passing the sample through Sephadex G25 (purchased from Sigma-Aldrich). SYBR Gold used for staining RNAs in acrylamide gels was purchased from Life Technologies. All other chemicals and solvents were purchased from Sigma-Aldrich and used as received unless stated. Characterization Techniques. 1 H NMR, 13 C NMR, 13 C DEPT135, heteronuclear multiple-bond correlation spectroscopy (HMBC), and heteronuclear single quantum coherence (HSQC) spectra were recorded on a Bruker Avance 500 MHz spectrometer using deuterated chloroform (CDCl3) as the solvent. Quantitative 13C NMR spectra were collected overnight on a high-resolution Bruker Avance 900 MHz spectrometer fitted with a cryoprobe and using an inversegated decoupling pulse (zgig30) program with a recycle delay of 19.6 s to ensure full relaxation of all nuclear spins between experiments. 5212

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Gel permeation chromatography-multi angle laser light scattering (GPC-MALLS) chromatograms were collected on a Waters GPC system equipped with an RI detector and a Wyatt 8 angle DAWN MALLS detector. The polymer was eluted at 1 mL/min in tetrahydrofuran (THF). dn/dc for the polymer was calculated by measuring the refractive index of a series of polymer dilutions in THF and was found to be 0.067 mL/g. UV−vis spectra were collected using a Varian Cary 4000 UV−vis Spectrophotometer. The extinction coefficient of trithiocarbonate endgroup (13,553 M−1cm−1) was measured from a series of standard CTAalkyne solutions in acetonitrile (Supporting Information, Figure S2). The concentration of RNA samples was measured using a Nanodrop 2000c spectrophotometer at 260 nm. High-performance liquid chromatography (HPLC) of RNA samples was collected on a Waters HPLC system equipped with UV−visible detector. The samples (∼50 μL) were injected into a Gemini reversed phase C18 column (150 × 4.6 mm, 110A) and run at acetonitrile gradient 1−15% in buffer KHCO3 pH 9 for 1 h. The detector was set up at 260 nm to monitor the progress of the separation. Polyacrylamide gel electrophoresis (PAGE) was performed on miniProtean Tetra Cell equipped with a PowerPac Basic power supply at 100 V for 2 h in TBE 1X buffer. Hand-cast gels used in this study were 1 mm thick. The visualization of the SYBR Gold-stained gels was carried out on a Gel Doc EZ System. Synthesis of Hyperbranched Polymer (HBP). PEGMA (3.7 mL, 8.42 mmol), CTA-alkyne (696 mg, 1.73 mmol), AIBN (27 mg, 0.17 mmol), EGDMA (385 μL, 2.04 mmol) and TFEA (30 μL, 2.34 mmol) were dissolved in 12 mL THF and sealed in a 25 mL round-bottom flask fitted with a septum. The mixture was purged with argon for 15 min in an ice bath, heated to 70 °C in an oil bath, and allowed to react for 24 h (98 mol % PEGMA conversion). Upon completion of the reaction, the product was precipitated in hexane three times then dried under vacuum. The resulting viscous oil was dissolved in water, transferred into dialysis tubing (SnakeSkin 10k MWCO) and dialyzed against water for 2 days. The dialyzed solution was lyophilized to yield the pure HBP which was subsequently analyzed by GPC-MALLS, NMR (1D and 2D experiments), and UV−vis spectrophotometry. The trithiocarbonate end-group was removed using a standard aminolysis procedure. In a typical experiment, HBP (200 mg, 0.0095 mmol, 0.0476 mmol end-group) was dissolved in 2 mL CDCl3. Ethanolamine (14.4 μL, 0.2381 mmol) was added to the solution. The reaction was stirred at room temperature for 24 h. The crude product was analyzed with 1H NMR and 13C NMR. MTS-Capped HPB. End-group aminolysis was performed in the presence of methylmethanethiosulfonate (MMTS) using the following procedure: HBP (200 mg, 0.0095 mmol, 0.0476 mmol end-group) and MMTS (90 μL, 0.9524 nnol) were dissolved in 2 mL acetonitrile. Ethanolamine (14.4 μL, 0.2381 mmol) was added to the solution. The reaction was stirred at room temperature for 24 h. The product was purified and characterized using the same procedure as described for the standard HBP outlined previously. RNA−HBP Conjugation. Unprotected thiol sense strand (ssRNA, 0.085 nmol/μL) was reacted with methyl disulfide functionalized HBP (0.028 nmol/μL) in KHCO3 buffer pH 9 at room temperature overnight. The reaction was analyzed and purified by HPLC and further analyzed on 15% PAGE-Urea. The conjugate was hybridized with antisense strand in HEPES buffer pH 7.5 and subsequently analyzed on 15% PAGE.

theory predicts that all chains will be terminated by either the R or Z group of the RAFT agent, three different methods were used to quantify the number of end-groups per HBP, including quantitative 1H NMR, 13C NMR, and UV−vis spectroscopy. An absolute molecular weight (Mn) for the polymer was determined to be 21 kDa with the molar mass dispersity equal to 1.6 as measured by GPC-MALLS analysis, while the Mn of each linear chain that makes up an arm of the hyperbranched polymer was calculated using the three spectroscopic methods. The results are reported in Table 1. Table 1. Comparison between Monomer Feed Ratio and the Resulting Repeat Unit Ratio after the Purification of the HBP

feed ratio (mol/mol) observed ratioa (mol/mol)

PEGMA (p)

TFEA (q)

EGDMA (r)

CTA-alkyne (x or y)

4.9 7

1.4 1.5

1.2 2

1 1

a Obtained from the quantitative 13C NMR spectrum of the HBP using eq 2

Diagnostic peaks from the 1H NMR spectrum shown in Figure 1 allows determination of individual chain lengths by comparison of end-group resonances with those assigned to monomer repeat units. Thus, integration of the methylene peaks arising from the alkyne end-group (∼4.5 ppm, A), TFEA repeat unit (4.2−4.4 ppm, B), and PEGMA repeat unit (3.8−4.2 ppm, C) allows calculation of the number of end-groups per HBP using eq 1 (Mn (1H NMR) of 5,200 with 4 end-groups per HBP). M n(1H NMR) =

A B (MW PEGMA) + (MW TFEA) C C + MW CTAalkyne

(1)

1

While H NMR provides a simple methodology for estimating the number of end-groups per polymer, some discrepancies arise using this approach due to poor resolution of the diagnostic peaks in the spectrum. Thus, the HBP was analyzed using quantitative 13C NMR of the polymer, where it is expected that the greater chemical shift dispersion of 13C NMR will overcome the problems with overlapping peaks observed in the 1H NMR experiments. In order to achieve quantitative 13C NMR, the delay time between pulses was set to 19.6 s such that all carbon nuclear spins with long relaxation times were fully relaxed prior to the next acquisition. In addition an inverse-gated decoupled pulse sequence was applied to avoid artifacts arising from the nuclear Overhauser effect.30,31 Finally, in order to collect the data in a relatively short time frame, the spectrum was collected on a high resolution 900 MHz spectrometer fitted with a cryoprobe. A typical resulting quantitative 13C NMR spectrum of the HBP is presented in Figure 2. The 13C NMR spectrum provides a number of important characteristic signals arising from both the Z- and R-groups of the RAFT agent on the polymer (note, many of these peaks were not distinguishable in the 1H spectra). The ratio of the integrals of the peaks due to the trithiocarbonate end-groups (220.8 ppm; Zgroup) and the CH of alkyne end-groups (75 ppm; R-group) is approximately 1.01. This indicates that both the α and ω endgroups are well-preserved during the polymerization and purification steps. Table 1 summarizes the feed ratio and the observed monomer ratio in the final polymer, as measured by 13C NMR. The number of repeat units (PEGMA, TFEA and EGDMA) per branch (10.5 units) was calculated using eq 2 and

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RESULTS AND DISSCUSION Synthesis of Polymer and End-Group Analysis. The synthetic procedure employed in this report involves random coupling of monomers in a statistical manner to form a lightly branched material. While the use of RAFT agents allows some control over the number of monomers that are coupled into a single chain, the incorporation of branching monomers (EGDMA) leads to intrinsic dispersity in structure arising from development of the hyperbranched polymers.9 Since RAFT 5213

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Figure 1. 1H NMR spectrum of HBP in CDCl3. The inset shows the expanded region of the spectrum where the methylene groups on PEGMA (p4) and TFEA (q4) are observed, along with the methylene end-group adjacent to the alkyne-terminus (y18). A to D are the integral values of the corresponding peaks. A detailed signal assignment of the spectrum can be found in the Supporting Information.

solutions of CTA-alkyne at a series of concentrations to determine the extinction coefficient of the compound. Using the assumption that the extinction coefficient of the CTA-alkyne and macro-CTA are the same, the Mn of the polymer was determined (Mn(UV−vis) as 4710 Da with 4.4 end-groups per HBP) (see Supporting Information, Figure S2 for detailed calculation). This was comparable to the value determined by 1H NMR. Table 2 shows a comparison of Mn calculated using the four different methods. While each method has its own advantages and disadvantages, end-group analysis using a quantitative 13C NMR spectrum provides more detailed information on the HBP structure, and potentially a more accurate determination of Mn can be obtained since it is the only method that allows direct analysis of individual resonances in the polymer. Importantly, it is imperative that an accurate measure of the end-group concentration can be determined for these polymers, since siRNA is attached to the polymer by postsynthesis modification of these groups (see Scheme 1). Distribution of Fluorinated Segments in the HBP. For imaging applications, the distribution of fluorinated segments throughout the hydrophilic polymer structure is of utmost importance for improving imaging properties.16,32,33 In order to develop a detailed understanding of the sequence of fluorinated units within the branched structure, HSQC and HMBC experiments were performed, as these techniques utilize the superior resolution of 13C NMR to determine near-neighbor couplings. The 1H NMR spectrum of the HBP shows a small, broad resonance centered at 4.7 ppm. While the broad nature of this peak suggested that it arose from protons attached to the polymer (and hence likely not an impurity), it could not be assigned directly from initial investigation of the polymer structure and the integrated intensity of this peak was smaller than expected for main-chain peaks. The HSQC spectrum in Figure 3 below indicates that this peak has a one-bond heteronuclear correlation with a carbon peak at 48 ppm

found to be higher than the theoretical number of repeat units (7.5 units), however this can most likely be attributed to loss of low molecular weight oligomeric material during the purification process leading to a higher apparent degree of polymerization. The Mn (13C NMR) was calculated using eq 3 and found to be 4160 Da which equates to five end-groups per polymer. This was comparable to the Mn calculated by 1H NMR. 13C NMR provides additional structural information due to observation of peaks which can be assigned to branching units (EGDMA) allowing determination of the branching content in the polymer (not directly resolved using other techniques). The degree of branching was calculated using eq 4 and was found to be 17.4 mol %. x=A=B=K≈1 y=C=G≈1 p = E/A = F /A ≈ 7 q = H /A = I /A ≈ 1.5 r=D−E≈2

(2) 13

where A to K are integral values of characteristic peaks in the C NMR in Figure 2. M n(1C NMR) = p(MW PEGMA) + q(MW TFEA) 1 + r(MW EGDMA) + MW CTAalkyne 2 (3)

⎛ ⎞ r degree of branching = ⎜ ⎟100% ⎝ p + q + r + x⎠

(4)

UV−vis provides an alternative method for calculating the number of end-groups, since the characteristic maximum absorbance of trithiocarbonate end-group at 306-312 nm provides information about the concentration of trithiocarbonate groups in solution. A calibration curve was constructed from 5214

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Figure 2. (A) Quantitative 13C NMR spectrum of HBP. The peaks at 220.8 ppm (x13) and at 75 ppm (y20) are due to the carbon of the trithiocarbonate ω end-group and the methine group at the α end-group, respectively. A to K are the integral values of the corresponding peaks. (B) Expanded region of the 13C NMR spectrum.

be assigned to the CH backbone of TFEA units immediately adjacent to the ω end-group of the polymer (or the Z-group of the RAFT agent). Quantification of the number of TFEA units adjacent to ω end-groups was achieved by 1H and 13C NMR analysis. Analysis of the 1H NMR integrals indicates that the terminal monomer group at the ω-end is exclusively the TFEA repeat unit, since the integral ratio of CH2O(y18) to CH (q2′) ≈ 2 (calculated from the ratio of C/D in the 1H NMR in Figure 1). Further analysis of the 1H NMR spectrum suggests that 69 mol % of TFEA (calculated from the ratio of 2D/B 100%) units are located next to the ω end-group, while 13C NMR analysis indicates this value is slightly higher at 73 mol % (calculated from the ratio of [((integral at ∼ 48 ppm)/(integral at ∼ 123 ppm))] in the quantitative 13C NMR). From these two methods of analysis, we suggest that their position next to the end-groups imparts

(delineated by the dotted line in Figure 3). In addition, according to DEPT-135 13C NMR (Supporting Information, Figure S6), this peak in the 13C NMR spectrum is attributed to a tertiary carbon, indicating that the broad signal in the proton spectrum arises from a methine group. To further examine the origin of this resonance, HMBC was performed to identify near neighbor atoms within a 4-bond distance. The spectrum reveals that there is a three-bond heteronuclear correlation between the broad proton signal at 4.7 ppm and the carbons of the trithiocarbonate end-group at 220.8 ppm (Figure 4). Furthermore, these −CH− protons also show correlation to carbonyl groups of TFEA repeat units at 170 ppm. The observed heteronuclear couplings are highlighted by the dotted lines in the HMBC spectrum and shown by arrows in the structure shown in the inset of Figure 4. In order for both of these couplings to be present, the proton peak at 4.7 ppm (q2′) must 5215

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group functionality of the polymers, postmodification for attachment of siRNA and other gene therapies was now possible. Although it is generally considered a mild process for removing trithiocarbonate end-groups, aminolysis of poly[(meth)acrylate] RAFT end-groups may lead to side-reactions that cause low yield of the desired product.34 It is well-known that without the addition of suitable capping agents, thiols generated from this reaction can undergo intra- and/or intermolecular disulfide reactions. For the HBPs reported in this manuscript, an excess of at least 5 equiv of ethanolamine is required to achieve quantitative cleavage of the trithiocarbonate end-groups in a 24 h reaction. However, under these conditions it was observed that the concentration of TFEA units in the final polymer also decreased. We attributed this to the vulnerability of TFEA ester groups to strong nucleophilic attack. To verify that this was indeed the mechanism of degradation of the TFEA, an HMBC spectrum of the aminolysis products was obtained after 1 day ethanolamine treatment of HBP in CDCl3, and indicates the formation of trifluoroethanol (signal at F1= 125 ppm; F2 = 3.85 ppm) side-products (Figure 5). While previous authors have suggested that thiol-backbiting to form a thiolactone species can occur upon aminolysis of poly[(meth)acrylate] RAFT endgroups,34,35 our data does not show any indication of this occurring. However, the HMBC spectra do show a 2-bond correlation that corresponds to a C12 thioamide byproduct at (F1 = 199 ppm; F2 = 3.81 ppm) and (F1= 199 ppm; F2 = 3.2 ppm). This signal would most likely arise following amine-

Table 2. Molecular Properties of the HBP method Mn HBP (g/mol) Mn per branch (g/mol) end-groups per HBP

H NMRa

UV−visb

− 5200

− 4710

− 4160

21 000 −

4

4.4

5

−

1

13

C NMRc GPC−MALLSd

a

Calculated using eq 1 from methylene peak ratio of PEGMA (p4), TFEA (q4), and alkyne end-group (y18). bCalculated using the extinction coefficient of trithiocarbonate end-group (13 553 M−1 cm−1). cCalculated using eq 2 from carbon peak ratio of PEGMA (p7), TFEA (q5), EGDMA (r3) and trithiocarbonate end-group (x13). dGPC-MALLS in THF (dn/dc = 0.067). Note: due to overlapping methylene peaks of PEGMA and EGDMA in the 1H NMR spectrum, the contribution of EGDMA in Mn(1H NMR) measurement is neglected. This makes the molecular weight measured by 1H NMR slightly larger than those measured by UV−vis and 13C NMR.

significantly enhanced molecular mobility to these moieties and may be one reason why these materials show good 19F MR signal. In our previous studies we demonstrated that this class of HBP can be successfully used for in vivo 19F MRI with short scan times, typically less than 10 min.16,15 A necessary requirement for this performance is high segmental mobility of the fluorinated groups reducing the strength of the dipolar couplings and hence leading to long transverse relaxation times. End-Group Reduction and Subsequent Modification of the HBP. Upon developing a strong understanding of the end-

Figure 3. HSQC spectrum of the HBP in CDCl3. A cross-peak (emphasized by the dotted lines) at coordinate F1 = 48 ppm; F2 = 4.75 ppm indicates a direct one-bond correlation between a proton at 4.75 ppm and a carbon at 48 ppm. 5216

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Figure 4. HMBC spectrum of HBP in CDCl3. The spectrum shows 2-bond correlation (5):Cx13−Hq2′ between the proton of methine groups at 4.75 ppm and the carbon of trithiocarbonate end-groups at 220.8 ppm. (1):Cq1−Hq2′, (2):Cq5−Hq4, (3):Cq3−Hq4, and (4):Cq3−Hq2′ (4) correlations provide supporting evidance that the trithiocarbonate end-group is directly adjacent to a TFEA unit.

Figure 5. HMBC spectrum of HBP crude products following aminolysis in CDCl3. The signal at coordinate (x1,x2) indicates a 2-bond correlation between methylene protons at 3.8 ppm and fluorinated carbons at 120 ppm arising from trifluoroethanol byproduct (A). Signals at coordinates (y3,y2) and (y1,y2) correspond to 2-bond correlations between methylene protons at 3.8 ppm and carbonyl groups at 200 ppm and between methylene protons at 3.2 ppm and carbonyl groups at 200 ppm. This suggests the formation of the thioamide byproduct (B).

proportional to the number of fluorine atoms present. Therefore, to minimize this side reaction, we performed the aminolysis in the presence of MMTS capping agent. MMTS was chosen for its high reactivity toward thiol/thiolate (rate constant toward thiolate anion of β-mercaptoethanol (BME) = 3.1 × 106 M−1 s−1).36 This is of the order of 100 times more reactive than the commonly utilized DPDS capping agent (DPDS rate constant

induced reduction of the TFEA ester group (see the structures in Figure 5). Upon further investigation, it was shown that greater than 75 mol % of the TFEA units were reduced during a 24 h aminolysis reaction. For the synthesis of theranostics proposed in this research, such a high loss of 19F signal is unacceptable for imaging purposes, since the imaging intensity in 19F MRI is directly 5217

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toward thiolate anion of BME = 5.6 × 104 M−1 s−1).37 Furthermore, MMTS not only protects the generated thiols against polymer−polymer coupling reactions, but the methanesulfonic acid byproduct (pKa = −1.9)38 can also neutralize excess ethanolamine, therefore reducing the cleavage of TFEA units during the reaction (Scheme 2). Indeed, in the presence of Scheme 2. Products Formed during Aminolysis of the HBP (A) without Thiol-Capping Agent and (B) in the Presence of MMTS

Figure 6. HPLC chromatogram detected at 260 nm of (A) thiolprotected single-stranded RNAs as purchased (46.1 min), (B) disulfidelinked ssRNA dimers (32.2 min), and (C) HBP/thiol ssRNA reaction (35.6 min).

analysis of the HPLC purified fraction at 35.6 min (Supporting Information, Figure S8) further indicates the formation of ssRNA-HBP conjugates as evidenced by a shift in elution of the conjugate compared to free ssRNA. Double-stranded RNA-HBP (dsRNA-HBP) conjugates were formed by heating of an equal number of moles of antisense RNA with an equal number of moles of purified ssRNA-HBP conjugate in duplex buffer (pH 7.5). To verify that the conjugates are indeed formed via a disulfide cleavable linkage, the dsRNAHBP conjugates were incubated in 0.2 M DTT for 10 min at room temperature. The nondenaturing PAGE (15% acrylamide) (Figure 7, line 3) shows the release of dsRNAs in a strong

MMTS (20 equiv), 78.6 mol % TFEA units remained after 24 h aminolysis using the same conditions as that described above (cf. 25 mol % in the absence of the capping agent and 35.7 mol % in the presence of DPDS). See Supporting Information, Figures S3−S5, for detail 1H NMR spectra and TFEA quantification. To further characterize the polymer product following aminolysis, 1H NMR was performed. A new peak at 2.4 ppm (Supporting Information, Figure S4) was assigned to methyl protons of methyl disulfide functional groups on the HBP. The ratio of the integrals of the peaks due to the methyl disulfide and the methylene of the alkyne end-group was found to be 1.5, indicating a quantitative conversion of trithiocarbonate to methyl disulfide groups. In addition, UV−visible analysis (Supporting Information, Figure S7) confirmed the removal of 96 mol % trithiocarbonate groups after the aminolysis. RNA−HBP Conjugation. The ultimate goal of modifying the ω end-group with MMTS was to facilitate the conjugation of thiol-modified therapeutic genes to the HBP.39 Scheme 1B shows the synthetic approach for stepwise attachment of the double-stranded RNAs to the HBP. Clearly, understanding the efficacy of this step requires detailed knowledge of the end-group structure as described above. Controlling the number of therapeutic genes attached to the carrier is one of the most challenging aims in designing an effective gene delivery system. In this study, we utilized an siRNA:HBP feed ratio of 3:1 (mol/ mol) in order to achieve conjugation of three RNA units to each HBP. Thiol-terminated sense strand of the siRNA was purchased for direct attachment to the MMTS-terminal HBPs. HPLC analysis (Figure 6) of the product of reaction of the single strand RNA and the HBP after overnight incubation at room temperature shows that 90% of the ssRNA was conjugated to the HBP, indicating that on average, 2.7 RNAs were successfully conjugated to each HBP (Figure 6C). In addition, ssRNA− ssRNA dimers (Figure 6B) were not detected in the reaction mixture, suggesting that in this experiment the thiol-exchange reaction shows preference toward the formation of the ssRNAHBP conjugates. Denaturing PAGE (15% acrylamide, 8 M Urea)

Figure 7. Polyacrylamide gel electrophoresis of native dsRNAs. Lane 1: hybridized thiol-protected dsRNAs. Lane 2: hybridized dsRNA-HBP conjugates. Lane 3: hybridized dsRNA-HBP in 0.2 M DTT. Gel was made of 15% acrylamide, run at 100 V for 2 h, and stained with SYBR Gold.

reducing environment. Thus, our synthetic approach has allowed us to develop a thorough understanding of the number of endgroups per polymer, as well as control the addition of siRNA via disulfide-cleavable linkers for gene therapy while maintaining the integrity of the 19F groups for MRI. 5218

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CONCLUSIONS We have demonstrated the design of a theranostic, PEG-based hyperbranched polymer for gene therapy and developed an in depth understanding of the structure of the polymer using wellestablished spectroscopic methods such as 1D and 2D NMR and UV−vis. In addition, we have revealed that the majority of fluorinated units within the branched structure are adjacent to the ω end-group, suggesting that this polymer has great potential to be developed as a molecular imaging agent for 19F MRI. Our thorough characterization of these materials has allowed us to further develop the HBPs for conjugation of therapeutic genes, making this polymer an ideal candidate for in vivo theranostics. Importantly, we have identified optimized routes for conjugation of biomolecules utilizing the RAFT end-group, without affecting the molecular integrity. Further tests on the effect of linker groups on in vivo stability of the conjugate, as well as the ability to incorporate different targeting ligands on the HBP are currently underway.

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ASSOCIATED CONTENT

* Supporting Information S

NMR spectrum of HBP, UV−vis spectrum profile, 1H NMR spectra of crude product, methyl disulfide functionalized HBP, and HBP after aminolysis, 13C NMR and 13C DEPT 135 spectra of HBP, UV−vis spectra of purified HBP, and polyacrylamide gel electrophoresis of denatured ssRNAs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(K.J.T.) E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge discussion with Dr Daniel Coles. The authors acknowledge the Australian Research Council (ARC) for funding (K.J.T., FT110100284, DP140100951; A.K.W., DP110104299). A.A. holds an Endeavour Postgraduate Scholarship. This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australia’s researchers. This research was conducted and funded by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036).

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REFERENCES

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dx.doi.org/10.1021/ma501196h | Macromolecules 2014, 47, 5211−5219