Design of Functional Nanoparticles for Intractable Disease Therapy

Abstract

Protein affinity reagents are widely used for basic research, diagnostics, and disease therapy. Antibodies and their fragments are known as the most common protein affinity reagents. They specifically and strongly bind to target molecules and inhibit their functions. Thus, antibody drugs have increased in the recent two decades for disease therapy, such as cancer. These strong protein–protein interactions are composed of a nexus of multiple weak interactions. Synthetic polymers that bind to target molecules have been developed by the imitation of protein–protein interactions. These polymers show nanomolar affinity for the target and neutralize their functions; thus, they are of significant interest as a cost-effective protein affinity reagent. We have been developing synthetic polymer nanoparticles (NPs) that bind to target peptides and proteins by the inclusion of several functional monomers, such as charged and hydrophobic monomers. In this review, the focus is on the design of synthetic polymer NPs that bind to target molecules for disease therapy. We succeeded in neutralization of toxic peptides and signaling proteins both in vitro and in vivo. Additionally, linear polymers were modified on a lipid nanoparticle surface to improve polymer biodistribution. Our recent findings should provide useful information for the development of abiotic protein affinity reagents.

1. PROTEIN AFFINITY REAGENTS

Our biological activities are supported with many protein–protein interactions (PPIs), including antibody–antigen, enzyme–substrate, and ligand–receptor interactions.¹⁾ Protein affinity reagents that bind to target molecules are able to change our biological activities through inhibition of these PPIs. Thus, they are used for disease therapy, diagnostics, and research. Antibodies and their fragments are the gold standards for inhibition of PPIs.²⁾ Although they specifically and strongly bind to target molecules, their high production cost and low reproducibility are still significant concerns. In nature, biomacromolecules recognize their binding target via the many weak complementary interactions, such as hydrogen bonds and hydrophobic and electrostatic interactions (Fig. 1a). Additionally, typically, PPIs are larger than 1000 Ų and involve more than 20 amino acid contacts.^3,4) Synthetic polymers, such as dendrimers,⁵⁾ linear polymers,^6–8) and polymer nanoparticles (NPs),^9,10) have the potential to work as protein affinity reagents by mimicking protein–protein interactions¹¹⁾ (Fig. 1b). For example, the Schrader group designed methacrylamide-based copolymers that bind to a specific epitope of enzyme.^12,13) The Haag group prepared sulfate-functionalized dendrimers for the inhibition of selectins in vivo.^14,15) The Shea group synthesized poly N-isopropylacrylamide (pNIPAm)-based NPs that bind to immunoglobulin G (IgG), snake toxin, and lipopolysaccharide.^16–19)

Fig. 1. Design of Synthetic Polymer Nanoparticles (NPs) by the Imitation of Protein–Protein Interaction

(a) Schematic image of the antigen–antibody interaction and (b) Schematic image of the antigen–nanoparticle interaction. (Color figure can be accessed in the online version.)

2. SYNTHETIC POLYMER NPS THAT BIND TO AND NEUTRALIZE TARGETS

To demonstrate that NPs bind to target molecules and neutralize their functions like an antibody, melittin, honeybee venom, was used as a target toxic model. Melittin is a positively charged amphiphilic peptide composed of 26 amino acids. Melittin makes a pore on the cellular membrane after binding to the cell surface.²⁰⁾ Because melittin is composed of neutral, hydrophobic, and positively charged amino acids, N-isopropylacrylamide (NIPAm, based monomer), N-tert-butylacrylamide (TBAm, hydrophobic monomer), N,N′-methylenebisacrylamide (Bis, cross-linker), and acrylic acid (AAc, negatively charged monomer) were used for the preparation of the NPs. In the beginning, NPs were prepared by molecular imprinting technology.^21,22) We found that inclusion of both negatively charged and hydrophobic monomers into the NPs is important for the induction of high melittin affinity. Inclusion of only negatively charged or hydrophobic monomers into NPs is not sufficient for the generation of a high melittin affinity. The optimized imprinted NPs showed a high melittin affinity and inhibited hemolysis induced by melittin in vitro. Additionally, the NPs did not show any affinity for plasma proteins, such as albumin and fibrinogen. To demonstrate the potential of the synthesized NPs in vivo, the NPs were intravenously injected into melittin-treated mice 20 s after melittin injection. The melittin biodistribution significantly changed after intravenous injection of the NPs. Melittin spread in the entire body after its intravenous injection; however, large amounts accumulated in the liver after the NP treatment.²³⁾ These results indicated that the NPs captured melittin in the bloodstream of living mice after intravenous injection. Additionally, more than 60% of the mice were dead after melittin injection with 24 h; however, all mice survived after the NP treatment, indicating that the NPs not only captured melittin but also neutralized it in the bloodstream. This result indicated that NPs have potential as a synthetic antibody.

3. SYNTHESIS OF POLYMER NPS THAT BIND TO AND NEUTRALIZE A TARGET WITHOUT MOLECULAR IMPRINTING TECHNOLOGY

Because the molecular imprinting method for the preparation of NPs that have a high target affinity uses the target molecule as a disposable material, the price of the imprinted NP will not be cheaper than that of the template. Thus, NPs that have a high target affinity need to be prepared without imprinting for mass production. To prepare non-imprinted NPs, we optimized the functional monomer percentage. We found that although a slight decrease in the binding affinity of the NPs for the target was observed compared with that of the imprinted NPs, non-imprinted NPs still had a high affinity for the target.²⁴⁾ Non-imprinted NPs captured melittin in the bloodstream and improved the survival rate of the melittin-treated mice. Thus, we found that non-imprinted NPs also work in vivo, and thus, non-imprinted NPs were used in the subsequent experiment.

4. ANTI-CANCER THERAPY WITH ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) PLASTIC ANTIBODIES

Melittin has a relatively simple structure because of the negatively charged amino acid-deleted peptide. However, nature proteins, such as signaling proteins, form a more complicated structure. To demonstrate whether NPs that have a high affinity for these signaling proteins can be prepared or not, we used vascular endothelial growth factor 165 (VEGF₁₆₅) as a target signaling protein. It is well known that VEGF is secreted from cancer cells and increases tumor growth by creating angiogenic blood vessels.^25–27) Bevacizumab (Avastin^®), a monoclonal antibody for VEGF, has made significant contributions to cancer treatment.^28,29) However, these treatments are very costly and impose a burden on patients and health care systems.³⁰⁾ Thus, “anti-VEGF NP” will be an attractive protein affinity reagent for cancer therapy. For the development of anti-VEGF NPs, we focused on the fact that VEGF has two different binding domains, a receptor-binding domain and a heparin-binding domain.³¹⁾ It is well known that heparin is highly sulfated,³²⁾ suggesting that inclusion of sulfated or heparin-mimicking monomers into NPs is important for the preparation of NPs that have a high VEGF affinity. NPs were prepared with NIPAm, TBAm, Bis, and a heparin-mimicking monomer (3,4,6 trisulfated N-acetylglucosamines monomer (3,4,6S-GlcNAc)) or 2-acrylamido-2-methylpropane sulfonic acid (AS) by modified precipitation _’polymerization³³⁾ (Fig. 2a). Although AS-containing NPs did not show any affinity for VEGF, 1.7% loading of the 3,4,6S-GlcNAc monomer into the NPs showed a high affinity to VEGF₁₆₅. An increase in the 3,4,6S-GlcNAc monomer percentage did not increase the affinity for VEGF but caused a decrease. Additionally, a decrease in the TBAm percentage from 40 mol% to 20 or 0% decreased the VEGF affinity, indicating that optimization of both 3,4,6S-GlcNAc and the hydrophobic monomer percentage is important for the generation of a high VEGF affinity. The optimized NPs did not show any affinity to heparin-binding domain-deleted VEGF (VEGF₁₂₁), indicating that the NPs bind to the heparin-binding domain of VEGF₁₆₅.

Fig. 2. Synthetic Polymer NPs That Bind to and Neutralize VEGF

(a) Functional monomers for the NP synthesis. (b) Inhibition of VEGF-dependent cell growth by NPs. HUVECs were incubated with NPs and VEGF₁₆₅ (20 ng/mL) for 48 h after preculturing the cell without growth factors and serum for 12 h. Then, viable cells were determined by MTT assay. Significant differences: * p < 0.05 and *** p < 0.001 versus VEGF₁₆₅ alone. (c) Schematic image of the VEGF–NP interaction. (d) Inhibition of tumor growth by the intravenous injection of NPs. Tumor-bearing mice were intravenously injected with PBS or NPs (5, 10, 20, or 40 mg/kg) at 5, 7, 9, and 11 d after tumor implantation. Significant difference vs. PBS: * p < 0.05, ** p < 0.01, and *** p < 0.001. (Color figure can be accessed in the online version.)

It is known that VEGF interacts with the receptor (VEGFR-2) that expresses on the cell surface.³⁴⁾ Then, phosphorylation of VEGFR-2 and cell growth enhancement are induced.³⁵⁾ Because anti-VEGF NPs bind to the heparin-binding domain of VEGF, binding of NPs to VEGF does not guarantee that NPs inhibit the VEGF–VEGFR-2 interaction. To demonstrate whether NPs inhibit the VEGF and VEGFR-2 interaction or not, human umbilical vein endothelial cells (HUVECs) were incubated with the NPs and VEGF. Then, VEGF-dependent phosphorylation of VEGFR-2 and cell growth was measured. As a result, NPs dose-dependently inhibited phosphorylation of VEGFR-2 and HUVEC growth (Fig. 2b). From these results, NPs not only were found to bind to VEGF₁₆₅ but also inhibited the VEGF–VEGFR-2 interaction (Fig. 2c). To demonstrate whether NPs inhibit tumor growth after intravenous injection through inhibition of the VEGF function, tumor-implanted mice were intravenously injected with NPs. Intravenous injection of NPs into tumor-implanted mice significantly inhibited the tumor growth (Fig. 2d) without any side effects.³⁶⁾ These results indicated that anti-VEGF NPs have potential as an attractive anti-cancer agent.

5. DESIGN OF POLYMER NPS THAT CAPTURE TARGET MOLECULES IN THE INTESTINES AFTER ORAL ADMINISTRATION

We showed that the synthesized NPs captured the target peptide and protein in the bloodstream of living mice; however, only few have reported that synthetic polymers specifically capture small molecules in the intestine of living mice. For proof of concept, we used indole, an aromatic low-molecular compound, as a model of a toxic small molecule.^37,38) Indole is synthesized from tryptophan by intestinal bacteria, such as Escherichia coli.³⁹⁾ After the adsorption of indole from the intestine, indole will be metabolized to indoxyl sulfate (IS), a uremic toxin, in the liver.^40,41) Although IS is excreted from the kidney in healthy subjects, it accumulates in the kidneys and causes damage in patients with chronic kidney disease.⁴²⁾ Thus, inhibition of indole adsorption from the intestine is one critical strategy. Because indole is a hydrophobic and aromatic compound, NPs were prepared with NIPAm, Bis and TBAm, N-phenyacrylamide (PAA), or 2,3,4,5,6-pentafluorophenyl acrylamide (5FPAA)⁴³⁾ (Fig. 3a). We found that an increase of hydrophobic monomer in the NPs also increased the indole capture rate. Additionally, incorporation of two hydrophobic monomers, TBAm and 5FPAA, increased the indole capture rate compared with a single hydrophobic monomer-containing NP. This suggested that TBAm made hydrophobic regions in the NPs and captured indole (Fig. 3b). However, 5FPAA captured indole via quadrupole interactions of the pentafluoro rings (Fig. 3b). These results indicate that incorporation of different binding modes in the hydrophobic monomer enhances the affinity of NPs for the target. The optimized NPs did not degrade by the digestive enzymes. Additionally, the orally administered NPs significantly inhibited orally administered [¹⁴C]-labeled indole, indicating that the NPs captured indole and inhibited indole adsorption from the intestines. These results indicate that synthetic NPs are attractive agents for capturing and inhibiting the adsorption of target molecules in the intestines.

Fig. 3. Design of NPs for Indole Capture

(a) Functional monomers for the preparation of indole-capturing NPs and (b) schematic image of the indole–NP interaction. (Color figure can be accessed in the online version.)

6. NEUTRALIZARION OF A TARGET PROTEIN IN THE BLOODSTREAM BY LIPOSOME ANTIBODIES

We have been developing synthetic polymer NPs that bind to and neutralize target molecules in the bloodstream. However, the NPs eliminated very shortly from the bloodstream after intravenous injection.³⁶⁾ The short circulation characteristic of NPs is a significant problem for long-term neutralization in the bloodstream. There are some examples in which the inclusion of polyethylene glycol into NPs reduced the affinity for plasma proteins⁴⁴⁾ and increased the circulation time.⁴⁵⁾ However, polyethylene glycol (PEG) incorporation into NPs may also reduce the target affinity. Thus, we focused on a lipid nanoparticle (LNP), which is a highly biocompatible drug delivery agent, to improve the polymer circulation time after intravenous injection. For the modification of the polymer to LNP, we synthesized a linear polymer (polymer ligands, PL) by reversible addition—fragmentation chain transfer polymerization and modified it on the LNP surface⁴⁶⁾ (Fig. 4a). Histones were used as the target toxic protein in the study. Histones usually packed in the nuclei; however, it was released into the bloodstream from the damaged cell. It is known that histones are major proteins for the induction of sepsis.⁴⁷⁾ Thus, histone neutralization reagents will be an attractive medicine for sepsis therapy. For the development of polymer-modified LNPs that have a high histone affinity, we used NIPAm, TBAm, and AAc as functional monomers and optimized the functional monomer percentage and polymer length. We found that a linear polymer composed of NIPAm : TBAm : AAc = 20 : 40 : 40 showed a high histone affinity. Additionally, the 100-mer polymer showed better affinity to the target protein than 30- and 1000-mer polymers. The polymer circulation time significantly increased after intravenous injection compared with that of the polymer alone. Surprisingly, the affinity of the polymer ligand for the target significantly increased upon modification onto the LNP surface because of the corporative effect of the modified ligands. Additionally, the histone neutralization effect in the living mice was significantly enhanced (Fig. 4b). We believe that these results will be useful for increasing polymer circulation time in vivo.

Fig. 4. Development of PL–NPs

(a) Schematic image of PL–NP. (b) Histone neutralization effect of PL-NPs. Mice were intravenously injected with histones (55 mg/kg), and 20 s later, the mice were intravenously injected with PBS, PL11 (3 µM/mouse as the PL concentration, purple), or PL–NP (2.6 µM/mouse (as the PL concentration). Then, the survival was monitored. Significant difference: * p < 0.05 vs. histone alone. Copyright Journal of Controlled Release.⁴⁶⁾ (Color figure can be accessed in the online version.)

7. CONCLUSION

In this report, I reviewed the design of synthetic polymers that exhibit high affinity for target peptide, protein, and small molecules. Protein affinity reagents can be used in several fields, including diagnostic, research, and medicine. In particular, considerable antibody therapeutics will be developed for disease therapy. However, the therapeutic cost may not be economical for many patients. Although there are numerous issues such as inflammatory induction, carcinogenesis, and metabolism of synthetic polymers for clinical applications, I strongly believe that synthetic polymers can replace antibodies in these fields as an economical protein affinity reagent in the future. Many synthetic polymers exhibiting high affinity and specificity toward the target have been fabricated by the imitation of nature protein–protein interaction. Furthermore, some synthetic polymers work in vivo. However, the number of in vivo applications of synthetic polymers is still considerably low. I hope this review will provide useful information for the in vivo application of synthetic polymers.

Acknowledgments

I would like to acknowledge Dr. Naoto Oku at Graduate School of Pharmaceutical Sciences, Teikyo University, Tomohiro Asai and Sei Yonezawa at Graduate School of Pharmaceutical Sciences, University of Shizuoka, and Kosuke Shimizu at Department of Molecular Imaging, Hamamatsu University School of Medicine. I wish to thank Dr. Yoshiko Miura and Yu Hoshino, Department of Chemical Engineering, Kyushu University, and Dr. Kenneth J. Shea, Department of Chemistry, University of California, Irvine. I also thank team “Polymer nanoparticles” (Saki Ariizumi, Chiaki Kiyokawa, Hiroki Tsuchida, Naoki Hayashi, Anna Okishima, Kosuke Shimizu, Ayaka Masuda, Yasuko Tempaku, Satoshi Hirano, Kazuhiro Saito, Hikaru Suzuki, Ikumi Yamauchi, Go Yasuno, and Hiroki Ochiai) and laboratory members at the Department of Medical Biochemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2020 Pharmaceutical Society of Japan Award for Young Scientists.

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