Letter pubs.acs.org/macroletters
pH-Responsive Transferrin-pHlexi Particles Capable of Targeting Cells in Vitro Adelene S. M. Wong,†,‡ Ewa Czuba,‡ Moore Z. Chen,‡ Daniel Yuen,‡ Kristofer I. Cupic,†,‡ Shenglin Yang,† Rebecca Y. Hodgetts,† Laura I. Selby,‡ Angus P. R. Johnston,*,‡,§ and Georgina K. Such*,† †
Department of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia § ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Australia ‡
S Supporting Information *
ABSTRACT: Targeting nanoparticles to specific cellular receptors has the potential to deliver therapeutic compounds to target sites while minimizing side effects. To this end, we have conjugated a targeting protein, holo-transferrin (holo-Tf), to pH-responsive polymers, poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) and poly(2-(diethylamino)ethyl methacrylate)-ran-poly(2-(diisopropylamino)ethyl methacrylate (PDEAEMA-r-PDPAEMA). These protein−polymer hybrid materials were observed to self-assemble when the pH is increased above the pKa of the polymer. We demonstrate that their response to pH could be tuned depending on the polymer constituent attached to holoTf. Importantly, the targeting behavior of these nanoparticles could be maximized by tuning the density of holo-Tf on the nanoparticle surface by the introduction of a (PDEAEMA-rPDPAEMA)-b-poly(ethylene glycol) (PEG) copolymer.
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nanocarriers.16−18 These polymers undergo a reversible change from a hydrophilic (charged) state to a hydrophobic (uncharged) state upon an increase in pH above their pKa. Upon internalization into acidic compartments within cells, these pHresponsive nanocarriers disassemble, releasing their therapeutic cargo.17 Our group has recently developed a pH-responsive PDEAEMA “pHlexi” nanoparticle system that disassembles at a pH of 6.8 at 37 °C. This system facilitates endosomal escape and cytoplasmic delivery of a fluorescent reporter cargo into the cytosol of cultured cells.19,20 Herein, we report a new hybrid system based on the charge shifting polymers PDEAEMA and PDEAEMA-r-PDPAEMA conjugated to a model biological targeting molecule, the protein holo-Tf. The two monomers, DEAEMA and DPAEMA, were chosen as the substituents on the nitrogen have been shown to allow tuning of the pKa of these polymers.21 We demonstrate that the conjugates are able to self-assemble to form hybrid particles with transferrin on their surface. We also demonstrate the successful tuning of the density of transferrin on the surface of the particles through the introduction of (PDEAEMA-r-PDPAEMA)-b-PEG to the system (Scheme 1). It is important that the surface density of
he conjugation of biological molecules such as proteins or peptides to polymers has gained increasing attention in recent years.1−4 Polymers can be synthesized with highly desirable physical properties and are easier to handle compared to proteins or peptides. On the other hand, proteins and peptides possess complex biological responses such as receptor targeting,5−7 enzymatic activity, and structural control that polymers do not.1,8 Hence, the use of protein−polymer conjugates possessing desirable properties from both classes of materials may be key to designing “smarter” nanocarriers for use in medical and therapeutic applications. Developments in controlled radical polymerization (CRP) methods such as atom transfer radical polymerization (ATRP)9 and reversible addition−fragmentation chain transfer (RAFT)10 polymerization have enabled the synthesis of polymers with controlled molecular weight and architecture. This control over size, along with the ability to incorporate various end-group functionalities, allows polymers to be designed with precise characteristics. These approaches, combined with high efficiency covalent coupling techniques such as click chemistry, enable the synthesis of protein−polymer hybrids.11−14 A variety of other nonclick coupling techniques have also been used to synthesize protein−polymer hybrids.15 Charge-shifting polymers, such as those containing tertiary amines, have been widely used in the design of pH-responsive © XXXX American Chemical Society
Received: January 22, 2017 Accepted: March 1, 2017
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DOI: 10.1021/acsmacrolett.7b00044 ACS Macro Lett. 2017, 6, 315−320
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ACS Macro Letters Scheme 1. pH-Dependent Self-Assembly of Tf-pHlexi Hybrid 1:0 Particles and 1:20 Blend Particles
transferrin is tunable as a dense concentration of proteins has been reported to increase nonspecific binding to cells.22 There have been several protein−polymer conjugate systems reported using the temperature-responsive poly(N-isopropylacrylamide) (PNIPAM).23−25 However, to the best of our knowledge, this is the first investigation into the covalent conjugation of the pHresponsive polymers PDEAEMA and PDPAEMA to a biological molecule. We demonstrate that the pH-responsive nature of the polymer translates to the assembled hybrid nanoparticle, which can be tuned based on the composition of the polymer. Importantly, we also demonstrate that the targeting potential of these particles could be maximized by tuning the density of holo-Tf on the nanoparticle surface. We believe this hybrid nanoparticle system is an improvement over conventional polymeric nanocarriers as it is able to combine the pH-responsiveness of synthetic polymers with the targeting capabilities of holo-Tf. The pH-responsive polymers PDEAEMA and PDEAEMA-rPDPAEMA were both synthesized by RAFT polymerization using a RAFT agent containing an alkyne functional group on the R-terminal. The molecular weight of PDEAEMA and PDEAEMA-r-PDPAEMA was determined by 1H NMR analysis to be ∼37 kDa and ∼79 kDa, respectively. Holo-Tf was functionalized with azide groups by reacting the amines present on the surface of holo-Tf with a 12-PEG unit Nhydroxysuccinimide-azide (NHS-azide) linker. Electrospray
ionization time-of-flight (ESI-TOF) analysis of the modified protein showed the successful azide functionalization of holo-Tf (Figure S5). The azide-modified holo-Tf was dye-labeled with sulfo-cyanine5 NHS ester, reacted with either PDEAEMA or PDEAEMA-r-PDPAEMA using a copper(I)-catalyzed azide− alkyne click chemistry reaction, and purified using extensive dialysis to remove unreacted polymer, protein, and catalyst. To demonstrate the conjugation of holo-Tf to PDEAEMA and PDEAEMA-r-PDPAEMA, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to compare the electrophoretic mobility of the conjugate, unreacted polymer, holo-Tf, and an unreacted mixture of holo-Tf and polymer (Figure S6). Successful conjugation of protein to polymer was demonstrated by a different migration pattern to either polymer or protein. The results also indicate that any unreacted protein had been effectively removed. Tf-PDEAEMA and Tf-(PDEAEMA-r-PDPAEMA) 1:0 particles were synthesized by a simple dialysis procedure to increase the pH of the conjugate solution from pH 6 to pH 8. A total of 3.0 mg of Tf-PDEAEMA or Tf-(PDEAEMA-rPDPAEMA) was dispersed into 3 mL of phosphate-buffered saline (PBS) at pH 6 and dialyzed against PBS at pH 8 over 7 h in a 3.5 kDa molecular weight cutoff (MWCO) dialysis tubing. The particles were then transferred to a 300 kDa MWCO dialysis tubing and dialyzed against PBS at pH 8 for a further 24 h to remove any free-floating components (full experimental 316
DOI: 10.1021/acsmacrolett.7b00044 ACS Macro Lett. 2017, 6, 315−320
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ACS Macro Letters
values using DLS. Tf-PDEAEMA 1:0 particles remained stable from pH 8.0 to pH 7.5 (white unshaded region) but completely disassembled at pH 7.3 (dark shaded region) (Figure 2a). The
details in Supporting Information (SI)). We postulate that when the protein−polymer conjugates are dispersed into a solution greater than the pKa of the respective polymers, hydrophobic interactions drive the assembly of the polymer components into a hydrophobic core, while the holo-Tf remains on the surface of the particles forming a hydrophilic corona. We also designed a 1:20 system comprising one part Tf-(PDEAEMA-r-PDPAEMA) to 20 parts (PDEAEMA-rPDPAEMA)-b-PEG (∼92 kDa) (see SI for synthesis details). In this system, the holo-Tf and PEG components both remain on the surface, stabilizing the particles. This 1:20 system was chosen because we expected that the surface of the 100% Tf(PDEAEMA-r-PDPAEMA) system would be too densely packed with holo-Tf, potentially resulting in increased nonspecific association with cells. Hence, we chose a Tf(PDEAEMA-r-PDPAEMA) to (PDEAEMA-r-PDPAEMA)-bPEG ratio that would reduce the density of holo-Tf on the surface of the particles, yet still maintain significant targeting potential. The number of holo-Tf proteins per particle in the 1:20 system was estimated to be ∼160 (see SI for details). Future studies will investigate tuning the ratio of these components to determine the optimized density of holo-Tf on the surface of the particles. The intensity mean diameters of the particle systems were determined by dynamic light scattering (DLS) to be approximately 80 nm (Figures 1a, 1c,
Figure 2. pH disassembly profiles of (a) Tf-PDEAEMA and (b) Tf(PDEAEMA-r-PDPAEMA) 1:0 particles. The white unshaded region indicates particle stability, whereas the dark shaded region indicates disassembled particles.
pKa of PDEAEMA is reported to be 7.0−7.3,26 while the pKa of PDPAEMA is lower at 6.0.27 Therefore, we expected the Tf(PDEAEMA-r-PDPAEMA) 1:0 particles to disassemble at a lower pH. We observed that the Tf-(PDEAEMA-r-PDPAEMA) 1:0 particles were stable from pH 8.0 to pH 6.9 (white unshaded region) but completely disassembled at pH 6.7 (dark shaded region) (Figure 2b). The formation and disassembly of these Tf-pHlexi particles demonstrate that the pH-responsive nature of the respective polymers was preserved in the protein−polymer conjugate system. The Tf-(PDEAEMA-rPDPAEMA)/(PDEAEMA-r-PDPAEMA)-b-PEG 1:20 blend system showed similar pH-dependent disassembly behavior to the Tf-(PDEAEMA-r-PDPAEMA) 1:0 particles (Figure S9). To investigate the targeting function of the modified transferrin, we compared the association of Tf-(PDEAEMA-rPDPAEMA) 1:0 and Tf-(PDEAEMA-r-PDPAEMA)/(PDEAEMA-r-PDPAEMA)-b-PEG 1:20 particles with a human cell line (HEK 293A) transiently overexpressing the recombinant transferrin receptor (TfR) (Figure 3a). HEK 293A cells were chosen as they have low endogenous expression of transferrin receptors on their surface,28 while a superfolder green fluorescent protein-tagged TfR construct (sfGFP-TfR) was used to discriminate between transfected and
Figure 1. Intensity size distributions of (a) Tf-PDEAEMA and (c) Tf(PDEAEMA-r-PDPAEMA) 1:0 particles in PBS at pH 8. Cryoelectron microscopy (cryo-EM) images of (b) Tf-PDEAEMA and (c) Tf-(PDEAEMA-r-PDPAEMA) 1:0 particles. Scale bars represent 150 nm.
and S7). Cryo-electron microscopy (cryo-EM) images were obtained of the Tf-PDEAEMA 1:0, Tf-(PDEAEMA-r-PDPAEMA) 1:0, and Tf-(PDEAEMA-r-PDPAEMA)/(PDEAEMA-rPDPAEMA)-b-PEG 1:20 particles, which confirmed the size and polydispersity results obtained by DLS (Figures 1b, 1d, and S8). To assess if these Tf−pHlexi particles were able to disassemble within a biologically relevant pH range (from pH 7.4 in the bloodstream to pH 5−6.5 in endosomal compartments), we assessed their size and stability over a range of pH 317
DOI: 10.1021/acsmacrolett.7b00044 ACS Macro Lett. 2017, 6, 315−320
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Figure 3. (a) Schematic representation showing the targeting action of Tf-(PDEAEMA-r-PDPAEMA) 1:0 and 1:20 particles to the surface of HEK cells transfected with transferrin receptor (TfR) plasmids. The figure illustrates the specific targeting of 1:20 particles to Tf receptors and the nonspecific association of 1:0 particles to the surface of the cell. (b) Flow cytometry analysis (mean fluorescence intensity normalized to the brightness of 1:0 particles) of the association of Tf-(PDEAEMA-r-PDPAEMA) 1:0 and 1:20 particles with HEK cells that are either unmodified (TfR negative) (blue) or transfected with transferrin receptor (TfR) plasmids (TfR positive) (red). Tf-(PDEAEMA-r-PDPAEMA) 1:20 particles were added at a concentration of 2700 particles per cell (1.8 × 10−4 mg mL−1 polymer), and 1:0 particles were added at a similar polymer concentration of 1.9 × 10−4 mg mL−1. (c) Flow cytometry analysis (mean fluorescence intensity) of the association of Tf-(PDEAEMA-r-PDPAEMA) 1:20 particles with TfR negative (blue) or TfR positive (red) HEK cells at three different concentrations of 200, 2700, and 12 000 particles per cell.
detached for analysis by flow cytometry. Figure 3b shows the association of 1:0 and 1:20 particles to TfR positive (TfR+) versus TfR negative (TfR-) HEK 293A cells. The 1:0 particles showed binding to the TfR+ cells but also exhibited a high level of nonspecific association to the surface of the TfR- cells, suggesting that the high density of Tf on the surface of the particles masks targeting to the TfR+ cells. However, with a lower density of holo-transferrin on the surface of the particles (1:20 particles), we observe specific targeting of the particles to
nontransfected cells. The targeting behavior of the PDEAEMAr-PDPAEMA-based particle systems was assessed instead of the PDEAEMA particles due to their more biologically relevant pH transition. The Tf-(PDEAEMA-r-PDPAEMA) 1:0 particles and 1:20 particles were added at equivalent polymer concentrations to 6 × 105 HEK 293A cells that were transfected with sfGFPTfR expression plasmid or untransfected control cells. The cells were incubated with particles for 15 min in bovine serum albumin (BSA) containing media, before being washed and 318
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ACS Macro Letters the TfR+ cells. While the normalized fluorescence intensity (NFI) of the 1:20 particles targeted to TfR+ cells was lower than the NFI of the 1:0 particles, the ratio of TfR+ to TfR- cells for the 1:0 particles was 1.5, but the NFI ratio for the 1:20 system was much higher at 3.9. We then proceeded to incubate TfR+ and TfR- cells with a range of 1:20 Tf particle concentrations, 200, 2700, and 12000 particles per cell, and studied their association behavior by flow cytometry (Figure 3c). The concentrations of the 1:20 Tf particles were determined using a nanoparticle tracking analysis software, which measures the scattering of individual particles. This also confirmed particle stability at the concentrations and conditions of the flow cytometry experiments. Results indicate that at concentrations of ∼200 and ∼2700 particles per cell we observe specific targeting of the particles to TfR+ cells, but at a concentration of ∼12 000 particles per cell, there was a high amount of nonspecific association observed. This shows that a lower concentration of particles minimizes nonspecific association to the surface of the cells. The results of these experiments collectively demonstrate that the Tf-pHlexi particles were able to retain the targeting capability of the Tf component in particle form, confirming that the process of synthesizing the particles had a limited effect on the biological activity of the protein, which is an essential factor in the design of conjugate materials. In summary, we have demonstrated the conjugation of a model protein, holo-transferrin to the pH-responsive polymers, PDEAEMA and PDEAEMA-r-PDPAEMA. The Tf-polymer conjugates self-assembled into uniform particles of ∼80 nm in diameter upon changing the pH of their environment from pH 6 to pH 8. We also demonstrate that it is possible to tune the density of holo-Tf on the surface of the particles by introducing a (PDEAEMA-r-PDPAEMA)-b-PEG component to the particle synthesis. We showed that the pH-responsive nature of the polymers was translated to the particles, where the pH of particle disassembly was tuned depending on the polymer constituent attached to holo-transferrin. Importantly, we demonstrated that the Tf-pHlexi particle system was capable of delivery to Tf receptors on the surface of cells, indicating the preservation of biomolecule functionality throughout the synthesis of the nanoparticles. This work provides a framework for the conjugation of pH-responsive polymers to sophisticated biological molecules such as highly engineered peptides for subsequent assembly into particles. The ability to combine the properties of both polymeric and biological materials allows for the design of materials with superior capabilities over traditional polymeric nanocarriers for drug delivery.
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Georgina K. Such: 0000-0002-2868-5799 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Australian Research Council through the Future Fellowship Scheme (FT120100564 - GKS and FT110100265 - APRJ) and the Centre of Excellence in Convergent Bio-Nano Science and Technology (APRJ).
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00044. Detailed experimental and characterization procedures, as well as additional data referred to in the text (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
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[email protected]. ORCID
Angus P. R. Johnston: 0000-0001-5611-4515 319
DOI: 10.1021/acsmacrolett.7b00044 ACS Macro Lett. 2017, 6, 315−320
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