Rational design of semiconducting polymer brushes as cancer theranostics

Abstract

Photonic theranostics (PTs) generally contain optical agents for the optical sensing of biomolecules and therapeutic components for converting light into heat or chemical energy. Semiconducting polymer nanoparticles (SPNs) as advanced PTs possessing good biocompatibility, stable photophysical properties, and sensitive and tunable optical responses from the ultraviolet to near-infrared (NIR) II window (300–1700 nm) have recently aroused great interest. Although semiconducting polymers (SPs) with various building blocks have been synthesized and developed to meet the demands of biophotonic applications, most of the SPNs were made by a nanoprecipitation method that used amphiphilic surfactants to encapsulate SPs. Such binary SP micelles usually exhibit weakened photophysical properties of SPs and undergo dissociation in vivo. SP brushes (SPBs) are products of functional post-modification of SP backbones, which endows unique features to SPNs (e.g. enhanced optical properties and multiple chemical reaction sites for the conjunction of organic/inorganic imaging agents and therapeutics). Furthermore, the SPB-based SPNs can be highly stable due to supramolecular self-assembly and/or chemical crosslinking. In this review, we highlight the recent progress in the development of SPBs for advanced theranostics.

1. Introduction

Nanotheranostics integrates diagnostics and therapeutics in a nanosystem and is promising for precision medicine.^1–11 Photonics-based nanotheranostic platforms combining molecular imaging and molecular therapy are extremely attractive in achieving early and fast detection of disease, synergistic personalized therapy, and real-time monitoring of the therapeutic effect during treatment.^12–28 Among the photonic nanoplatforms, the organic self-assembled semiconducting polymer nanoparticles (SPNs) can be designed at the molecular level to possess various photonic and chemical properties, thereby satisfying the requirements of rapidly developing biomedicine.^29–34

Semiconducting polymers (SPs) are organic electronic materials with π-conjugated polymer backbones composed of double bonds or aromatic rings.^35–40 In SPs, the overlapping p-orbitals form highly delocalized electrons, thereby showing excellent optoelectronic properties.^41–47 Compared with small dyes and pigments, SPs have a more stable structure and higher light-harvesting ability.³⁴‘^48–61 The electrons in SPs can be excited by a single photon or two photons and then undergo radiative transition and nonradiative vibrational relaxation.^61–63 The decay of electronic energy in SPs leads to fluorescence (FL), phosphorescence emission, and heat production.^55,64–75 The electronic energy in SPs can also alter ambient chemicals such as the transfer of O₂ to singlet oxygen ¹O₂. Thus, SPs are developed as versatile contrast agents for FL, two-photon, Raman, and photoacoustic (PA) imaging of cells and living tissues and therapeutic platforms for photodynamic therapy (PDT), photothermal therapy (PTT), and photoactivated theranostics.^76–93

Given the restriction of synthesis solvents of SPs (which can only be non-polar and high-boiling-point organic solvents), SPs with long alkyl chains are used as alternatives. Therefore, at an early stage, the most reported SPNs for biomedical applications were developed through nanoprecipitation of multifunctional amphiphilic surfactants.^61,94–101 However, there are obvious problems and obstacles to further develop SPNs. Binary SP micelles formed by nanoprecipitation usually exhibit weakened photophysical properties, especially after the conjugation of large biomolecules to the SPN surface, which may lead to off-target effects of SPNs and cause side effects, in the fast blood circulation. Furthermore, through nanoprecipitation, SPs naturally precipitate fast in water and show severe aggregation, which causes low FL emission, weak photodynamic properties, and low sensitivity to outside biomarkers.^62,102–104 To advance the development of SPs in biomedicine, further chemical modification of SPs is vital to develop SP-based theranostic platforms with high efficacy, multifunction, and safety.

With great efforts on chemical synthesis, rigid SPs have been covalently modified with functional groups, such as amphiphilic quaternary ammonium salt, polyethylenimine (PEI), polyethylene glycol) (PEG), or reactive groups. The functional groups linked on the backbone of long SPs exhibit a brush-like structure, which is often called a SP brush (SPB).^105–111 The SPB possesses several merits in constructing theranostics. First, SPB has a typical amphiphilic structure. Thus, fabricating SP nanoparticles in water is convenient via self-assembly. Moreover, the formed SPNs are relatively stable because of their natural hydrophobicity and covalent conjunction between the SP and the hydrophilic brush. Second, the brush can change the aggregation state of the SP, which improves the optical-electronic and nano-structural properties compared with surfactant-encapsulated SPs. Third, the photonic properties of SPNs can be improved by introducing photoactivable linkers with therapeutics into SPBs for photoactivated theranostics. In this review, we comprehensively summarize the recent development of SPBs in cancer theranostics (Fig. 1).

Fig. 1.

Schematic illustration of SPBs for advanced theranostics. The therapeutics include gene therapy, chemotherapy, photothermal therapy (PTT), enzyme-based therapy, photodynamic therapy (PDT) and radiotherapy. The diagnostics include magnetic resonance imaging (MRI), two-photon imaging, photoacoustic imaging, fluorescence imaging, afterglow imaging and computerized tomography (CT).

2. Enhanced optoelectronics of SPB theranostics

To demonstrate how the hydrophilic brushes can affect the optical properties and biophysical features of SPs after SPN fabrication. Xie et al. grafted different numbers and lengths of hydrophilic PEGs onto the backbone of diketopyrrolopyrrole (DPP)-based SPs (called SPAs) (Fig. 2).⁶² The SPAs were synthesized by using a typical two-step method, wherein the SP backbone precursor with reactive groups was first obtained via Suzuki coupling polymerization. Then, the click reaction was conducted to obtain PEG brushed SPBs. After the synthesis of the four SPBs, namely, SPA1PEG2, SPA1PEG5, SPA2PEG2, and SPA2PEG5, their size, FL, and PA effect were systematically compared. After self-assembly in water, only SPA1PEG5 and SPA2PEG5 showed spherical morphology with a hydrated diameter of 20 nm. According to the study, the nanosize of SPNs increased due to the increased hydrophobicity caused by a shorter and fewer number of PEG. The backbone of the SPB is the donor-acceptor (D–A) structure, which is highly electron-delocalized. Therefore, after self-assembly in water, the aggregation with strong π–π stacking resulted in an absorption redshift and fluorescence quenching. SPA2PEG2 showed the strongest FL intensity and the highest quantum yield (4.31%) compared with SPA2PEG5 (3.13%), SPA1PEG2 (2.77%), SPA1PEG5 (2.73%), and SPN1 (1.02%). However, the PA properties of the four SPNs were almost the same. The results showed that photo-induced thermal decay is the main process of DPP-based SPNs, and the PEG brushes can effectively decrease the aggregation to enhance SPB emission. For in vivo imaging, the strong π–π stacking and hydrophobic properties of SP chains warrant the stability and safety of SPNs.

Fig. 2.

Illustration of the structure of DPP based SPs and SPBs, and the two methods of SPN preparation: nanoprecipitation and self-assembly. The obtained SPNs showed the fluorescence enhancement effect by the addition of PEG brushes to the DPP backbone. Reproduced with permission.⁶² Copyright 2017 Wiley-VCH.

As SPBs can directly self-assemble into small and stable nanostructures, a plethora of SPB-based activatable probes were developed for cancer detection. Hypoxia is a typical feature of tumor tissues resulting from rapidly proliferating tumor cells and oxygen consumption.^112–116 The local O₂ is reported to be around 4% and even decreases to 0% at the center of the tumor. Thus, developing O₂ probes for precise cancer imaging in vivo is urgently required. Huang et al. developed a fluorescence/phosphorescence dual-emissive SPB through the copolymerization of O₂-sensitive phosphorescent platinum(II) porphyrin and O₂-insensitive fluorine (PF-P) (Fig. 3).¹¹⁷ The synthesized SPB can self-assemble into ultrasmall nanoparticles (PF-Pdots) with a dynamic light scattering (DLS) size of 5 nm. The FP-Pdots showed excellent sensitivity for O₂. The advantage of PF-Pdots over platinum(II) porphyrin was that O₂ detection can be ratiometric due to the fluorescence resonance energy transfer (FRET) effect from fluorine to platinum(II) porphyrin. The PF-Pdots allowed the reversible imaging of the O₂ content in living cells cultured at different O₂ concentrations via I_red/I_blue ratio values. The authors also found that the phosphorescence lifetime of the PF-Pdots (33.7 μs in N₂ and 9.9 μs in air) towards oxygen showed a significant prolongation. Thus, the FP-Pdots can be applied for time-resolved photoluminescence lifetime imaging and time-gated luminescence imaging of intracellular O₂ levels and can detect tumor hypoxia in mice. In the organic-metal complex based SPB, the platinum(II) porphyrin can be replaced with phosphorescent Ir(III) complexes on the SP backbone, which could retain the ratiometric oxygen sensing ability (phosphorescence lifetimes were 0.6 μs in N₂ and 0.41 μs in air) and show highly efficient generation of ¹O₂ under light irradiation. With the help of cationic brushes, the Ir(III) based SPNs can penetrate through the cell membrane into the cytoplasm and induce cellular apoptosis, which showed great potential for in vivo PDT (Fig. 4).¹¹⁸

Fig. 3.

(A) Scheme of the self-assembly process of cationic D–A SPBs. (B) Schematic of the electronic energy level of the fluorene and Pt(II) porphyrin moieties in SPBs and the oxygen sensing mechanism of the SPN. Reproduced with permission.¹¹⁷ Copyright 2015 Wiley-VCH.

Fig. 4.

(A) Structure of the Ir(III) complex based cationic SPBs. (B) TEM images of the self-assembled SPNs. (C) Mechanisms illustrating the oxygen sensing capability and photodynamic effect of the SPNs. Reproduced with permission.¹¹⁸ Copyright 2014 Wiley-VCH.

As cancer cells in hypoxia gradually and inevitably metastasize, exploring valid imaging probes and methods for imaging metastatic tumors is essential. Afterglow luminescence is a kind of self-illumination after exogenous activation of energy, such as light energy and radioactive energy. Afterglow luminescence imaging is an emerging paradigm of self-illuminating imaging, which shows a relatively high signal-to-background ratio (SBR) over conventional photoluminescence imaging.^55,119–122 SP, an organic optical polymer, is an excellent candidate for afterglow imaging because it has multiple reaction sites in the backbone.⁴⁶ Poly(phenylene vinylene) (PPV) was reported to react with singlet O₂ to form unstable dioxetane units. The chemical defects of dioxetane units can spontaneously break down to release photons in several minutes for afterglow imaging. Recently, Pu et al. found that the amphiphilic PPV brush-formed SPN is more efficient for afterglow imaging than the surfactant-encapsulated SPN (Fig. 5).¹²³ Three PPV structures with various side chains, PPV-PEG1, PPV-PEGL, and MEHPPV, were then synthesized and characterized. The NIR afterglow luminescence emission of SPs can be obtained by the FRET effect from PPV to silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NCBS), a hydrophobic NIR dye. Among the three SPs, PPV-PEG1 showed strong afterglow luminescence but was unable to encapsulate NCBS efficiently, due to the lack of hydrophobic parts. The PPV-PEGL with hydrophobic building blocks is relatively appropriate for loading NCBS to show NIR afterglow luminescence. Compared with the completely hydrophobic MEHPPV-based SPNs (PPVP) (34 nm), PPV-PEGL could encapsulate NCBS to form smaller sized SPNs (SPPVN) (24 nm). SPPVN and PPVP had similar UV absorption spectra; however, SPPVN (51%) had a 2.1-fold higher ratio of emission intensity at 780 nm than PPVP at 590 nm (24%). Furthermore, under the same mass concentration, the afterglow imaging intensity of SPPVN was 1.3-fold higher than that of PPVP. The authors explained that the enhancement of FRET and the afterglow effect were attributed to the more effective contact between the PPV segments and NCBS within SPPVN than that in PPVP.

Fig. 5.

(A) Structures of PPV based SPBs: PPV-PEG1 and PPV-PEGL. (B) Schematic illustration of the different preparation methods of SPPVN and PPVP. (C) DLS results and TEM images of SPPVN and PPVP. Reproduced with permission.¹²³ Copyright 2018 Wiley-VCH.

Recently, light in the NIR II region (950–1700 nm) has been developed for PA imaging because NIR II light has high tissue penetration and low background absorption for enhancing spatial resolution and increasing high SBR compared with NIR I (700–950 nm) PA imaging.^124–126 Given these merits, the NIR II laser is also suitable for deep-seated tumor PTT. Also, the NIR II PTT agents usually have higher photothermal conversion efficiency than NIR I PTT agents.^127–131 Thus, designing a robust SPB for biomedical applications in the NIR II region is interesting. The NIR II PA image-guided NIR II PTT of the tumor requires SPBs with a super narrow bandgap for absorbing photonics beyond 1000 nm and excellent photothermal stability for enduring continuous laser irritation. Most recently, Yin et al. synthesized NIR II SPBs with strong electronic D–A units formed by the Stille coupling polymerization of alkyl bromide-based quinoxaline and thiophene derivatives (Fig. 6).¹³² Such a strong electronic transfer validated the SP by extending the absorption spectra to the NIR-II window. The NIR II SPB was finally obtained through the Cu-catalyzed “click” reaction of conjugating multi-PEG to the side chain of the SP. Through self-assembly, the formed SPNs had a size of less than 100 nm with good stability in 10% fetal bovine serum. Owing to the strong absorbance at 1064 nm, the SPN demonstrated significant improvement in PA imaging under the NIR II laser compared with that under the NIR I laser. For example, at a depth of 1.5 cm, the PA imaging was still clear under the NIR II channel but was barely visible under the NIR I channel. After systemic administration, the NIR II PA can precisely indicate the location of the tumor via the contrast enhancement of SPNs, and mouse tumors can be effectively ablated by light.

Fig. 6.

(A) Chemical structure of the quinoxaline based SPB and the illustration of the SPB self-assembly for fabricating NIR-II PA imaging and PTT agents. (B) Photograph of the SPN solution shown by using the color of straw yellow. Reproduced with permission.¹³² Copyright 2019 Elsevier.

3. Multifunctional SPB theranostic platform

Multifunctional cancer theranostics includes a versatile imaging modality and individualizing therapeutic methods in one system for obtaining tumor images, continuous monitoring, and efficient drug delivery. Multiple model imaging can overcome the differences in biodistribution and selectivity that currently exist between imaging contrasts, and individualizing therapeutic agents have the potential for precision medicine.¹³³ Progress in nanotechnology stimulates the development of novel multimodal imaging contrast agents, thereby reinforcing the precise localization of the diseased tissue.¹³⁴ Currently, several multimodal imaging platforms have been developed, such as functionalized gold nanoparticles or self-assembly for positron emission tomography (PET)/computerized tomography (CT)¹³⁵ and PA/PET¹³⁶ and semiconducting perylene diimide platforms for PA/FL/PET imaging.¹³⁷

A SPB with NIR absorbance usually possesses two imaging capabilities of PA and FL. With careful modification of the brush, more imaging modalities can be introduced, such as PET and MR imaging. Fan et al. synthesized a narrow bandgap SPB (1.56 eV) with a backbone demonstrating strong absorbance at 808 nm for PA imaging and PTT after excitation (Fig. 7).¹³⁸ The SPB can also emit long-wavelength photons at 1100 nm, a kind of fluorescence in the NIR II window (1000–1700 nm), providing unparalleled advantages for the visualization of histology and pathology with higher spatial resolution and deeper tissue penetration than conventional NIR FL imaging. The SPB is based on PEG and carboxyl groups, which provides excellent water solubility and metal chelatability, respectively. When SPBs self-assembled into nanoparticles, the addition of gadolinium ions into the solution crosslinked SPBs to reinforce the stability of the SPNs and provided T₁-weighted MR imaging. After the SPB theranostics indicated the tumor location, PTT can be applied for tumor ablation.

Fig. 7.

(A) Schematic description of the multimodality imaging of PA, MRI and NIR-II FL for the guidance of PTT. (B) Structure of the multifunctionalized SPB and the SPN preparation through self-assembly and gadolinium ion chelation. Reproduced with permission.¹³⁸ Copyright 2019 Ivyspring.

Owing to the hydrophobic and rigid SP backbones, it is believed that the fluorescent SP can be fabricated as robust hollow nano-SP frameworks with cavities for drug loading and monitoring, which is similar to hollow mesoporous silica nanoparticles. This idea can be used to prepare a SP-based controlled release system. By utilizing the advantage of polymer science and nano-self-assembly technology, Wu et al. synthesized ultraviolet photo-crosslinkable SPBs based on the backbone structure of fluorene with oxetane groups and cationic photopolymerizable units on the side chains (Fig. 8). Due to the amphiphilic and degradable properties, poly(lactic acid-co-glycolic acid) acted as a porogen hybridizing with the SPB to co-self-assemble into nanoparticles by adjusting different weight ratios and then etching in hot alkaline solution after photo-crosslinking to form multicolor, water-dispersible SP nanocavities (PNCs). To demonstrate the essentiality of oxetane brushes for PNCs, the SP without oxetane groups was synthesized as the control structure. Unfortunately, SPNs without cavities were obtained. The PNCs demonstrated efficient delivery of small drugs, and their intracellular release can be monitored in real time by the fluorescence signal because of the energy transfer from fluorescent guest drugs to host SPs. Given that the pore size of PNCs is tunable, after loading amphiphilic PEI into PNCs, macromolecular small interfering RNA (siRNA) can be delivered for the gene silencing of cancer cells. The colorful PNCs are promising for biomedical imaging and drug delivery.¹³⁹

Fig. 8.

(A) Functional structure of the colorful SPB. (B) Schematic of the preparation of hollow SPNs for drug and gene loading. Reproduced with permission.¹³⁹ Copyright 2018 Wiley-VCH.

The SPB theranostics can also become three-dimensional nano-frameworks by coupling the polymerization of the spatially branched SP. Yang et al. reported carboxyl group-based hyper-branched SP (HSP) nano-frameworks, that were directly obtained via the Suzuki polymerization of monomer 2,2′-(2,7-dibromo-9H-fluorene-9,9-diyl)diacetic acid and tridirectional 1,3,5-tris(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene under the catalysis of palladium(0) (Fig. 9).¹⁴⁰ The directly obtained SPN was only 5 nm in size. Poly(maleic anhydride-alt-1-octadecene) C18-PMH-PEG was then used to modify the formed SPN for biocompatibility. The final nanoparticle showed a uniform and narrow size distribution at around 20 nm in water. Owing to the chelation of abundant carboxyl groups to metal ions, the SPN can be loaded with diagnostic radionuclide ^99mTc with a radiolabeling yield of approximately 84%. ¹³¹I can be labeled on the aromatic ring of SPs with a radiolabeling yield of approximately 56%. The radioactive SPN was used in mice for single-photon emission computed tomography (SPECT)/CT imaging and radionuclide therapy. With the help of 5,6-dimethylxanthenone-4-acetic acid, a tumor vascular disrupting drug, the radioactive SPN achieved remarkable slowdown and even halted the tumor growth of mice.

Fig. 9.

(A) Synthesis routes of 3D SPBs, and radiolabeling and PEGylation of the ultrasmall SPNs. (B) Schematic of the improved tumor EPR effect and therapeutic efficacy of SPNs with the help of a tumor-vascular disrupting drug, DMXAA. Reproduced with permission.¹⁴⁰ Copyright 2018 American Chemical Society.

4. Stimuli-responsive SPB theranostic platform

Theranostic nanoparticle accumulation in the tumor region relies on the enhanced permeability and retention (EPR) effect because of the leaky, irregularly shaped, and tortuous tumor vessels.^141,142 Stimuli-responsive theranostic platforms can interact with tumor microenvironment (TME)-associated biomarkers or external tumor stimulation to improve the EPR effect and enhance the efficacy of the theranostics.^143–146

4.1. TME responsive SPB theranostics

Active targeting strategies require the EPR effect to deliver the cargo to the tumor region and retain the payload for specific receptor/biomarker interactions via chemical modification of expensive targeting ligands/antibodies onto the nanoparticles. In contrast, the TME responsive self-assembly of SPNs can reinforce the EPR effect, which may be a simple and universal way to design efficient theranostics. Molybdenum (Mo)-based POM clusters, a kind of smart ultrasmall nanoparticles with self-adaptive electronic structures, have the ability of weak acidity-induced aggregation and NIR absorbance enhancement by reduction, which promotes the conversion of valence state of Mo(vi to v).¹⁴⁷ Given the advantages of SPB (e.g., optical tunable, self-assembling, and multiple binding sites on the brush), Chen et al. designed TME responsive POM based SPB theranostics (Fig. 10).¹⁴⁸ The abundant Mo-based POM clusters were introduced to the surface of SPNs via the co-self-assembly of SPBs and POMs in DMSO aqueous solution. In preparing synergistic hybrid phototheranostics, the maximum absorption of the SP macromolecule was first tuned to 808 nm to match the absorption of the transformed POM for effective PTT by tuning the ratio of DPP in Stille coupling polymerization. Then, the thiol groups were modified onto the brush for chelating POMs. After systemic administration to tumor-bearing mice, enhanced tumor accumulation of the SPB theranostics was demonstrated by both PET and amplified PA imaging. With the improved tumorous NIR absorbance in the 808 nm region, the laser was applied for tumor PTT.

Fig. 10.

(A) Structure of DPP based SPBs with abundant thiol groups in the brushes, and the hybrid SPB@POM showing the properties of acidity induced aggregation. (B) Schematic of the SPB@POM fabrication, and the tumorous pH/GSH-responsive SPB@POM for enhanced phototheranostics. Reproduced with permission.¹⁴⁸ Copyright 2018 Wiley-VCH.

TME is known to be oxidative. Wang et al. took advantage of the oxidative TME to rationally design redox responsive SPBs for cancer therapy. The TME-responsive SPB theranostic reversed the chemo-resistance of cancer. A water-soluble oligo(p-phenylene vinylene) (OPV) SP was modified using the brushes containing paclitaxel (PTX) units and thiol groups (Fig. 11).¹⁴⁹ After self-assembly, the small SPNs in water only showed a hydrated diameter of around 10 nm at a concentration of 2.5 μm, which was attributed to the hydrophobic interactions and π–π stacking between OPV and hydrophilic groups. The OPV-based SPNs can diffuse into cancer cells where the thiol groups on the brush could react with one another to cross-link the SPNs via the catalysis of reactive oxygen species (ROS). The phenomenon can also be confirmed via TEM. With the addition of H₂O₂ to SPNs, the size of SPNs increased obviously compared with that of the original SPNs. In cell experiments, after the internalization of the responsive SPNs, the theranostic SPNs showed a persistent effect on FL imaging after crosslinking. The chemotherapeutic effect of SPB theranostics also improved dramatically. The cellular toxicity results showed that the IC50 of SPNs for the A549 cell line was 0.33 nM, indicating a 124-fold decrease compared with that of PTX (41 nM). For the multidrug-resistant cancer cells (MCF-7m), SPNs also showed improved efficacy (6.7 μM) compared with that of PTX (30.2 μM). This significant therapeutic effect may be explained by the fact that SPNs can accelerate cellular microtubule fixation and induce cell apoptosis. In mouse experiments, the mouse tumor showed a persistent fluorescence signal even at 48 h post-injection of SPNs, which demonstrated that the responsive cross-linking effect boosted the retention effect of SPNs. Tumor growth was suppressed by SPN treatment and showed a better tumor inhibition rate (54%) compared with PTX (25%).

Fig. 11.

(A) Chemical structures of PTX drug derivatives by conjugating with pentaerythritol tetrakis(2-mercaptoacetate) and PPV brushes, respectively. (B) Schematic of self-assembly of the SPB drug and oxidation induced crosslinking. Reproduced with permission.¹⁴⁹ Copyright 2018 Wiley-VCH.

4.2. Photoactivated SPB theranostics

Most recently, the SPB photoactivated theranostic platform was developed and considered for precision medicine by taking advantage of the advanced optoelectronic properties of SPBs. After the accumulation of SPB theranostics in a tumor, photoactivated theranostics of SPNs was achieved with a proper laser through its selectively generated ultraviolet light for activating the SPN itself, electronic energy for sensitizing O₂ to ¹O₂ to cleave the ROS sensitive bond, and heat energy for disrupting supramolecular interactions. Furthermore, therapeutic cargo release can be realized through the PDT-induced hypoxia TME.¹⁵⁰ Herein, two aspects of the SPB photoactivated theranostics are discussed: photoactivation gene-based and chemo-based synergistic therapies.

4.2.1. Photoactivated gene-based theranostics.

Nucleic acid therapy holds promise in the treatment of cancer. For gene therapy, polynucleotides such as siRNA, aptamers, and plasmids are popular therapeutics that can alter gene expression at the transcriptional or post-transcriptional level, which may be effective for the treatment of cancer and other diseases. The success of gene regulation relies on its efficient delivery to the disease site. However, many obstacles affect the delivery of polynucleotides. For example, their large size and negative charge make internalization difficult due to the same negatively charged plasma membrane. The genes are also extremely easy to be degraded by in vivo enzymes. Thus, the delivery system of gene therapeutics is critical and should be carefully designed.^151–153

siRNA, sometimes called silencing RNA, is a kind of double-stranded non-coding RNA molecule with a length of 20–25 base pairs. siRNA is considered a therapeutic agent by operating within the RNA interference pathway.¹⁵⁴ To deliver the therapeutic siRNA to cancer cells, polymers or nanoparticles with a cationic group are preferred to bind the electronegative siRNA. However, the precise regulation of gene expression at designated tumor locations is essential to minimize off-target gene expression. Fabricating a delivery system of siRNA with the capability of non-invasive light-controlled gene release is desirable. Zhao et al. described a synergistic therapeutic strategy combining photocontrollable siRNA and PDT (Fig. 12).¹⁵⁵ In their theranostic system, the cationic brush in the polyfluorene brush, an electron-rich SP, was synthesized through the quaternary ammoniation of the abundant tertiary amine-functionalized SPB by photodegradable 2-nitrobenzyl-2-bromoacetate. The synthetic cationic brush can effectively bind the electronegative siRNA and achieve photo-induced charge variability to the zwitterionic brush for accelerating siRNA release under ultraviolet light. Furthermore, the fluorene-based SPB enabled the photosensitizer performance for PDT after ultraviolet light excitation. UCNPs were integrated into the SPB nanoparticles as a photo converter to provide an ultraviolet light source with the help of a 980 nm laser. The experimental results demonstrated the good stability of the hybrid SPB theranostics after siRNA loading. An excellent gene loading capacity of 1 mol NPs for 32.5 mol siRNA can be achieved. Upon 980 nm light irradiation, 80% of siRNA can be released in a TME-mimic environment. In vitro and in vivo experiments also demonstrated the great therapeutic efficacy of the photoactivated theranostics.

Fig. 12.

Schematic illustration of the preparation of the photoactivation gene delivery system and its function in biomedical applications. The mechanism of the photo-induced charge-variable cationic SPB is shown in the dashed box. Reproduced with permission.¹⁵⁵ Copyright 2017 Wiley-VCH.

Whole organic theranostics is preferred in clinical translation because of the potential toxicity of inorganic nanoparticles. Considering that two-photon SPBs can emit high energy light, researchers found an organic two-photon SPB substitution that can replace UCNPs to realize the charge convertibility of SPBs in the photocontrollable gene delivery system. Poly(phenylene ethynylene) PPE is an SP backbone that is capable of large two-photon cross-section absorption (Fig. 13).¹⁵⁶ The side brush was polymerized with photo-responsive and positively-charged chromophore 4,5-dimethoxy-2-nitrobenzyl 2-bromoacetate. Through time-resolved photoluminescence, significant FRET was effectively transferred from SPs to the photo-responsive side chains with 720 nm illumination, in which the two-photon-induced photolysis induced the structural change from cationic structures to zwitterions. The final self-assembled SPB gene delivery system showed the photoactivated release of siRNA in vitro, and 40% gene knockdown was achieved after irradiation.

Fig. 13.

Schematic of the chemical structures of two-photon activatable PPE based SPBs and the mechanism of the SPB for the photoactivation of siRNA release. Reproduced with permission.¹⁵⁶ Copyright 2019 American Chemical Society.

Photoactivated gene theranostics can also be achieved by the delivery of cytotoxic ribonuclease A (RNase A) to induce cancer cell death by degrading their intracellular RNA. Caging RNase A and preparing activatable RNase A are challenging while applying the cytotoxic RNA enzyme in cancer theranostics. The self-assembled SPB with a stabilized nanostructure and photodynamic properties is considered a desirable candidate for carrying the enzyme. As a proof-of-concept study, Li et al. fabricated a PEG-based amphiphilic photodynamic SPB covalently linked with an amine-based ¹O₂ cleavable linker, (Z)-2,2′-(ethene-1,2-diylbis-(sulfanediyl))diethanamine (Fig. 14).¹⁵⁷ After self-assembly, the amine on the SPN surface was modified with the caged RNase A via the amidation reaction. After 808 nm laser irradiation, the SPB theranostics demonstrated that the ¹O₂ generation not only induced the cleavage of the activatable linker to release caged RNase A but also gave rise to PDT for cancer. Given the increased H₂O₂ concentration in TME by PDT,¹¹⁹ the caged RNase A restored the enzyme activity for cancer-specific RNA degradation. The phototheranostics can not only kill the cancer cells but also downregulate the expression of metastasis-related proteins, including vascular cell adhesion molecule-1, hepatocyte growth factors, and metastasis-associated protein 2 to inhibit metastasis during cancer treatment.

Fig. 14.

(A) Schematic of the mechanism of the photoactivated synergistic SPN therapeutics including PDT and tumor cell RNA degradation. (B) Synthesis routes of the caged RNA enzyme and the photoactivated SPB. (C) SPN modification with the caged RNA enzyme on the surface. (D) Structure of the SPB and the control SPNs without enzyme modification. Reproduced with permission.¹⁵⁷ Copyright 2019 American Chemical Society.

Using the regularly clustered interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) is one of the most effective RNA-guided genome editing methods that is intensively developed and applied in the study of gene function, disease modeling, and disease therapeutics. The CRISPR/Cas9 genome editing system has revolutionized genome editing technology because of its relative simplicity and high efficiency and specificity.^158,159 In general, the efficient delivery of this system is critical to fully realize its editing function. Physical approaches such as electroporation and microinjection, and viral vectors are the current main delivery platforms for CRISPR/Cas9. However, the potential off-target effect of nonspecific accumulation in non-targeted tissues is easily overlooked, which can induce dangerous results such as genetic mutation. Local light regulation of CRISPR/Cas9 gene editing is a potential method to reduce the side effects. Nitrobenzene derivatives as photolabile caging groups were used for preparing a photoactivated CRISPR/Cas9 gene editing system.¹⁶⁰ However, the gene editing can be initiated by ultraviolet light, whose shallow tissue penetration limited the further application in vivo. Thus, NIR light for the local regulation of CRISPR/Cas9 gene editing is highly desirable.

In the previous studies by Chen et al., they fabricated DPP-based SPs that can be regulated with a 75% ratio to fluorine in the SP backbone, owing to strong absorption at 808 nm and excellent photothermal properties.¹⁴⁸ This DPP-based SP has excellent NIR II emission at around 1200 nm with the aggregation state in nanoparticles. An amphiphilic SPB was thus designed, in which the carboxyl-based electron donor fluorine was modified with PEI polymers and the electron acceptor DPP segments were modified with long alkyl chains and PEGs (M_w, 5000) (Fig. 15).¹⁶¹ The SPB can self-assemble in water to encapsulate dexamethasone (Dex), a glucocorticoid that binds with the nuclear glucocorticoid receptor to dilate the nuclear pores for boosting gene entering. The PEI on the SPN surface can wrap CRISPR/Cas9 cassettes via electrostatic interaction for genome editing. After accumulation in the tumor, the SPNs showed accelerated Dex release and gene endolysosomal escape after laser irradiation. The payload release of CRISPR/Cas9 cassettes allowed highly efficient genome editing. The advanced feature of this design is the visible and controllable genome editing in vivo under NIR-II FL guidance and NIR light stimulation, respectively. A tumor model was used to demonstrate that photocontrollable genome editing can be achieved in vivo. Fluorescence imaging of the tumor tissue showed decreased GFP fluorescence signals at 6 days post-injection. The deep sequencing data showed that the SPB gene editing system can achieve indel mutations at a frequency of ≈ 20%.

Fig. 15.

Schematic of the preparation of the SPN based CRISPR/Cas9 delivery system through SPBs encapsulating dexamethasone and binding CRISPR/Cas9 pDNA, and the photoactivation for intracellular genome editing. Reproduced with permission.¹⁶¹ Copyright 2019 Wiley-VCH.

Another strategy of photodynamic effect activated CRISPR/Cas9 genome editing was reported by Pu et al. They fabricated a photolabile SPB gene delivery system for CRISPR/Cas9 cassette loading (Fig. 16).¹⁶² The cassettes can be released through the specific photocleavage of the gene binder PEI brush on the SP background, which can realize controllable photo-regulated genome editing. The SPB macromolecular initiator was synthesized by monomer 1 (2,6-dibromo-4,4-bis(6-bromohexyl)-4H-cyclopenta[2,1-b:3,4-b′]dithiophene), monomer 2 (2,6-dibromo-4,4-bis(6-azido-hexyl)-4H-cyclopenta[2,1-b:3,4-b′]dithiophene), and monomer 3 (1,3-benzothiadiazole-4,7-bis[boronic acid pinacol ester]) through palladium-catalyzed Suzuki coupling polymerization. This kind of SP backbone showed outstanding photodynamic properties. The hydrophilic PEI brush and PEG were linked to the backbone through a thioketal ¹O₂-cleavable linker and by a direct click reaction, respectively. After self-assembling the SPB in water, the formed electrical SPNs can bind and condense genes onto SPNs through electrical interactions. The expression of the fluorescent GFPs in living cells by delivering inactive GFP genes that included plasmid nanocomplexes (Cas9 and sgRNA) was used to demonstrate the function of CRISPR/Cas9 gene editing. Under laser irritation, the thioketal was cut off by the generated ¹O₂ and then accelerated the release of the PEI binding CRISPR/Cas9 gene system to promote the gene function process.

Fig. 16.

(A) Schematic of the structure and self-assembly of the SPB with the linking of ¹O₂ cleavable PEI. (B) pSPN/CRISPR nanosystem for NIR laser controlled gene editing. Reproduced with permission.¹⁶² Copyright 2019 Wiley-VCH.

4.2.2. Photoactivated chemo-based theranostics.

In a photoactivable drug delivery system, the self-assembled SPB nanoparticles usually afford tunable physical and structural changes to respond to critical issues, such as photothermal and photo-induced chemical bond cleavage and photonics-induced change of TME, in the tumor region after systemic administration. Senthilkumar et al. reported a class of D–A Stenhouse adducts (DASAs), which showed a fast visible light (550 nm) response undergoing a change of properties in the physical structure from hydrophobic to hydrophilic forms (Fig. 17).¹⁰⁰ The DASA and the tumor-targeting molecule folic acid were synthesized as pendants in the PPV to form an amphiphilic photoactive SPB. Before 550 nm irradiation, the DASA showed a hydrophobic triene form, which is helpful for the self-assembly of the SPB. DOX and camptothecin can be encapsulated into SPNs during nanoprecipitation. The photoactivation of drug release can be achieved by a structural change of DASA from a hydrophobic form to a hydrophilic colorless ring-closed cyclopentenone form by laser irradiation. Because of the FRET effect between the DASA and the SP, the structural change and drug release can also be monitored via fluorescence enhancement at 535 nm. After the uptake of the SPB drug, confocal imaging of cancer cells showed significant CPT release in the cytoplasm and nucleus after laser irradiation, indicating the drug release.

Fig. 17.

(A) Chemical structure of PPV SPBs with photoactivated Stenhouse adduct brushes. (B) Illustration of the composition of the SPB drug loading system, and the cellular drug release accelerating upon 550 nm irradiation. Reproduced with permission.¹⁰⁰ Copyright 2018 Wiley-VCH.

Photo-regulating TME is also a promising approach to fabricate a photoactivated drug delivery system. During the application of PDT, the rapid ¹O₂ generation is accompanied by dramatically decreasing O₂ concentration in the tumor region.¹⁹ Thus, the amplified hypoxic TME induces the capability of the reduction enzyme system in the tumor, such as the well-known nitroreductases coupled to NADPH, which can reduce nitro groups to amino groups in the chemical structures. By utilizing this photo-induced hypoxia effect, Gu et al. designed photoactivated theranostics, in which a photosensitizer, a dithiophene-benzotriazole-based SPB, was conjugated with abundant hydrophobic hypoxia-sensitive 2-nitroimidazole (Fig. 18).¹⁶³ After nanoprecipitation with the help of amphiphilic polyvinyl alcohol, the hydrophobic SPB and DOX were co-self-assembled into nanoparticles to produce a photoactivatable synergistic therapeutic platform. The FL imaging signal from the SPB can be used to indicate the accumulation of the SPB theranostic platform. When the maximum accumulation was achieved, a laser was applied to irradiate tumors for ¹O₂ generation. Concurrently, the hypoxia sensitive SPB was reduced. The SPB became amphiphilic and promoted the dissociation of the platform to DOX simultaneously because of the hydrophilic properties of the reduced amino groups. The synergistic theranostics demonstrated good tumor inhibition compared with PDT.

Fig. 18.

(A) Schematic of the formation of the hypoxia-responsive SPB theranostics and its mechanism of photoactivation. (B) Photoactivation enhanced therapeutic efficacy by the PDT boosted DOX release from the SPB theranostics. Reproduced with permission.¹⁶³ Copyright 2016 Wiley-VCH.

The chemo drug conjugated to the nanoparticle surface can more easily stimulate release than the encapsulation inside the nanoparticle. Furthermore, the nanoparticle formed by one SPB structure through self-assembly is usually more stable than through multi-component nanoprecipitation. Cui et al. reported that a well-defined SPB structure can directly be self-assembled for cancer photoactivation chemo-based theranostics (Fig. 19).¹⁶⁴ The SPB was carefully covalently modified with a PEG brush composed of a bromoisophosphoramide mustard intermediate (IPM-Br) in the end through hypoxia-cleavable linkers. Notably, the SPB backbone demonstrated photodynamic and photothermal generation and NIR FL emission, which are valid for phototheranostics. The SPB can self-assemble into uniform nanoparticles of around 30 nm in size. The NIR FL imaging indicated the successful accumulation of SPB theranostics. After the hypoxia triggered by PDT, the IPM-Br was ready to be released by the cleavage of hypoxia-responsive bonds killing cancer cells by inducing their DNA crosslinking. The mouse tumor growth was significantly inhibited because of the synergistic photothermal, photodynamic, and chemotherapies.

Fig. 19.

(A) Chemical structure of the photoactivated prodrug SPB and the SPN preparation through self-assembly. (B) Schematic of the SPNs for photoactivated synergistic PDT and chemotherapy. Reproduced with permission.¹⁶⁴ Copyright 2019 Wiley-VCH.

Tumor-responsive release of cancer drugs is helpful to reduce side effects during treatment. Active imaging for monitoring drug release and guiding photoactivation therapy can increase the therapeutic efficacy. The multifunctional phototheranostics can readily be obtained by combining SPBs with inorganic nanomaterials. Yu et al. designed a smart theranostic system by coating amphiphilic SPBs, an polyelectrolyte-based zwitterionic photosensitizer, on the surface of hydrophobic upconversion nanoparticles (UCNPs, NaYF4:Yb and Tm@NaYF4) (Fig. 20).¹⁶⁵ The hypoxia-activated prodrug AQ4N was then loaded before coating the last layer of pH-sensitive Mn-Ca₃(PO₄)₂. The hybrid SPNs were dissociated to release hypoxia-activated drugs under an acidic TME and simultaneously triggering the MRI signal to indicate nano-accumulation and drug release. The combination of SPBs and UCNPs provides PDT under 980 nm laser irradiation. The PDT-induced hypoxia further activated the AQ4N for cancer PDT and combination chemotherapy.

Fig. 20.

Schematic of the fabrication of hybrid SPB based nanotheranostics and its dual-modal imaging guided synergetic PDT and chemotherapy. Reproduced with permission.¹⁶⁵ Copyright 2019 Elsevier.

5. Conclusion and outlook

In this review, we summarized the recent progress on the rational design of SPBs and the resulting SPNs for diagnostic and therapeutic applications. By carefully manipulating the D-A structure of the SP backbone, the photonic SP macroprecursor allows two-photon excitation and full-spectrum absorption and emission for biomedical applications such as FL imaging, PA imaging, PDT, and PTT. Multifunctional chemical groups were modified to the side of SPs for the fabrication of smart SPB theranostics. First, hydrophilic group modification makes SPBs eligible for self-assembly into stable SPNs with good water solubility and biocompatibility, which can be made possible mainly through a hepatobiliary route. After brush modification, the optical properties of the SPB were significantly improved compared with the initial SP. Second, the reactive groups on the brushes of SPs enable metal ion or metal nanoparticle chelation for multimodal imaging and therapy. Third, activatable linkers conjugated between SPs and functional groups result in a photoactivation therapeutic system for photocontrollable chemo drug or gene release and synergistic therapy.

Despite the significant progress in recent years, SPB theranostics is still at the laboratory stage. Promoting the development of SPBs so that it can be applied in the clinic is our final goal. First, imaging in the NIR II region is a promising technology for pushing forward SPBs for human use. Recently, through NIR II window imaging, indocyanine green was proven to be a guide in human liver tumor surgery and showed higher tumor-detection sensitivity, remarkable tumor-to-normal-liver-tissue signal ratios, and enhanced tumor-detection rate than NIR I FL imaging.¹⁶⁶ This is a huge opportunity to move the study of the NIR II-based SPB to clinical trials, especially for NIR II imaging-guided surgery with advanced properties of SPBs, such as ultrabright NIR II emission and photostability. With the development of the PA imaging system, NIR II PA images have demonstrated high spatial resolution and deep penetration depth. Until now, very few works have reported the activatable SPB PA imaging probe even though the SP demonstrates good NIR II PA imaging properties. Thus, the narrow band SPB is promising for smart NIR II PA probe design. Second, inorganic-organic hybrid SPB theranostics showed great potential in clinical translation as it can be modified with complementary imaging modalities. The key to developing the SPB hybrid for humans is to find multifunctional inorganic materials with low toxicity. Third, in vivo biodegradation is meaningful to decrease the long-term toxicity of SPB theranostics. The backbone and brushes of SPBs can be designed with cleavable bonds, which can slowly respond to oxidizing and reducing agents in vivo. By all accounts, efforts on the rational design of SPBs can accelerate the biomedical application of SPB theranostics in the future.

Biography

Zhen Yang

Zhen Yang obtained his PhD in Optical Engineering in 2017 from the Nanjing University of Posts and Telecommunications under the direction of Prof. Wei Huang and Prof. Quli Fan. During his PhD study, he joined Prof. Xiaoyuan (Shawn) Chen’s Lab at the National Institutes of Health (NIH) as a joint doctoral student from 2015 to 2017, after which he became a postdoctoral fellow in the same lab. His research interest focuses on the design and synthesis of multifunctional phototheranostics for multimodal imaging-guided synergistic therapy.

Ling Li

Ling Li received her BS degree in Pharmaceutical Engineering from Sichuan University in 2012 and her PhD in Pharmaceutics from Sichuan University in 2016. She joined Prof. Xiaoyuan (Shawn) Chen’s Lab at the National Institutes of Health (NIH) as a postdoctoral fellow in 2018. Her research interests mainly focus on gene therapy and immunotherapy.

Albert J. Jin

Albert J. Jin received his PhD in theoretical/mathematical physics from the University of Maryland at College Park in 1992, and then moved to NIH as a postdoctoral fellow in interdisciplinary biomedical research. He became a staff scientist in 2000. And since 2010, he has served as the Chief of the Nanoinstrumentation and Force Spectroscopy Section, within the Laboratory of Cellular Imaging and Macromolecular Biophysics, in the Intramural Research Program of NIBIB/NIH.

Wei Huang

Wei Huang received his PhD from Peking University in 1992. In 2001, he was appointed as a chair professor at Fudan University, where he founded and chaired the Institute of Advanced Materials (IAM). After that he became the vice president of Nanjing University of Posts & Telecommunications, and then the president of Nanjing Tech University. Now he is the deputy president of Northwestern Polytechnical University. His research interests include organic/plastic materials and devices, nanomaterials and nanotechnology, etc.

Xiaoyuan Chen

Xiaoyuan (Shawn) Chen received his PhD in Chemistry from the University of Idaho in 1999. He joined the University of Southern California as an Assistant Professor of Radiology in 2002. He then moved to Stanford University in 2004 and was promoted as an Associate Professor in 2008. In the summer of 2009, he joined the Intramural Research Program of the NIBIB as a tenured senior investigator and chief of the Laboratory of Molecular Imaging and Nanomedicine (LOMIN). His interests include developing molecular imaging tools for the early diagnosis of disease, monitoring therapy response, and guiding nanodrug discovery and development.