Qixuan Dai ,En Ren ,Dazhuang Xu ,Yun Zeng ,Chuan Chen ,Gang Liu

a Department of Pharmacy,Xiamen Medical College,Xiamen,361023,China

b State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics&Center for Molecular Imaging and Translational Medicine,School of Public Health,Xiamen University,Xiamen,361102,China

c Amoy Hopeful Biotechnology Co.,Ltd.,Xiamen,361027,China

Keywords:ICG Nanomedicine Self-assembly Tumor theranostics Tumor synergistic treatment

ABSTRACT As the sole cyanine dye approved by the Food and Drug Administration (FDA),indocyanine green (ICG) has been widely applied in solid tumor and metastases surgical navigation as well as malignant tissue destruction.The great performance of ICG benefits from excellent fluorescent properties in the near infrared region (NIR) and reactive oxygen species (ROS) or hyperthermia effects under irradiation.Nevertheless,some intrinsic limitations,such as unstable optical properties,concentration-dependent aggregation,proneness to photo-bleaching,and poor aqueous stability in vivo,severely restrict its further applications.To overcome these challenges,ICG-based nanodrugs(nano-ICG),as one kind of portfolio strategy,could bridge the efficient cargo-carriers and ICG molecules to artificially improve their existing performance.In this mini-review,we will focus on the developed engineering strategies for nano-ICG formulation synthesis and their desired performance in tumor tracing and eradication.A critical analysis of assembly mechanisms,excellent performance,and further improvement tactics will be examined.

1.Introduction

Indocyanine green (ICG) is an amphiphilic,inert,and non-toxic compound in physiological fluids which is composed of two hydrophobic polycyclic parts as well as sulfonic acid groups [1].As the only near-infrared (NIR) fluorescent dye introduced in clinical application and approved by the Food and Drug Administration (FDA),ICG has many excellent performances compared to other fluorescent dyes [2].For example,it has low toxicity,is quickly excreted by the liver into bile juice,and has a specific absorption maximum at the hemoglobin isosbestic point.In aqueous solutions,the maximum absorption peak of ICG monomer is approximately 800 nm.Meanwhile,its maximum emission value red-shifts from 810 to 820 nm in water to 830 nm in the circulating of blood [3].These specific optical characteristics prompting the ICG can be visualized through 5–10 mm of tissue via an appropriate imaging system.So far ICG has widely explored as a fluorescence(FL)and photoacoustic(PA)tomography imaging contrast agent for tumor surgical navigation and hepatic function study,especially in angiography to allow for the direct intraoperative visualization of the blood vessels in surgical procedures[4].Additionally,the specific reactive oxygen species (ROS) or hyperthermia effects that are motivated under external light source irradiation have laid the foundation as therapeutics for solid tumor destruction via photodynamic therapy(PDT)and photothermal therapy(PTT)as well as the sonosensitizer for sonodynamic therapy (SDT) [5,6].To date,more new capabilities of ICG are slowly being discovered,such as sentinel lymph node (SLN)detection,intraoperative angiography,visualization of colorectal liver metastases and hepatocellular carcinomas (HCCs),and so on [7–9].However,their major applications are in regard to solid tumor and metastases surgical navigation and malignant tumor destruction.

Despite the advantages mentioned above,the free ICG molecule suffers from several intrinsic issues in tumor tracing and eradication.The main drawback of ICG is non-specific bonding to the proteins in the circulation of the blood,their lack of targeting specificity for tumor lesions and the fast body clearance (2–4 min) in vivo.In addition,instability in water and aggregation properties induce self-quenching or low quantum yield that will reduce the sensitivity of the photosensitizer or sonosensitizer [1,3].The modification tactics and smart portfolio strategies for enhancing the existing performance of ICG molecules are urgently needed.

Up to now,a nanomaterial-based cargo delivery system has been a major trend for disposing of some significant challenges against small molecules,which is expected to improve the free molecules’ therapeutic efficacy and specificity [10].In fact,engineering nanocarriers have several advantages with an elaborate design.For instance,stimulating responsive nanomaterial can deliver molecular agents in a controllable manner,preventing premature leakage and achieving on-demand drug release (e.g.,light,temperature,ultrasound,magnetic field,and electric field)[11–14].Additionally,they can passively accumulate in tumor sites by an enhancing permeability and retention(EPR)effect or actively target tumor lesions with tumor-targeting ligands (e.g.,antibody,peptides,aptamers,folic acid) linkage [15].Furthermore,multiple decoration strategies for these nanocarriers will have the ability to alter their intrinsic characteristics,such as blood circulation,bio-distribution,and cellular uptake (e.g.,polyethylene glycol (PEG),liposomes,cell membrane vesicles(MVs),virus-like particles(VLP))[15,16].Of particular note is that as one kind of versatile drug carrier,the multiple components mentioned above could be loaded together with customization requirements as a portfolio strategy for achieving synergistic therapeutic effects (e.g.,chemotherapeutic drugs,therapeutic nuclides,antibodies,nucleotide drugs)[17].Therefore,a nanomaterial-based ICG delivery system (nano-ICG)must be a desirable strategy for optimizing ICG molecules to overcome the inherent poor performance and adding extra superior capacities[18].

Taken together,various ICG-based nano-formula strategies have been developed after unremitting efforts.Based on the interaction types of ICG molecules with the selected nanomaterials,two main categories of these nanomedicine-based portfolio strategies have been developed.The most common is functional nanomaterial(e.g.,some kind of inorganic and organic nanomaterial)like a“bullet shell”for ICG loading or encapsulating[1,3,19].The other category is hybrid nanoparticles in which ICG molecules themselves will be skeletal components of nano-ICG via self-assembling with various functional moieties[20].Different loading tactics have their own unique advantages and corresponding disadvantages.In terms of self-assembly nanomaterials,good loading capacity can be regarded as one kind of skeletal component of their carrier [21].Additionally,compared to the chemically synthesized carriers,the specific bioactive macromolecule (e.g.,proteins,nucleic acids,saccharides,lipid cholesterol)with low immunogenicity could interact with the ICG and reduce their own immunogenicity.

To date,several articles have reviewed the versatility of ICG for cancer theranostics in nanomedicine and nanotechnology research[1,3,22].This review will highlight these two most efficient modalities regarding respective advantages and disadvantages.As shown in Scheme 1,ICG can be delivered by nanocarriers (e.g.,mesoporous materials,polymeric nanocarriers,lipid-based carriers) and have innate ability for assembly with various ingredients (e.g.,proteins,peptides,metal ions,small molecules)through hydrophilic/hydrophobic action,electrostatic interaction and π-π stacking effect.In the end,some practical suggestions for the optimum combination strategy will be discussed.We hope this mini-review will attract more cooperative interest from different fields,such as chemistry,materials science,biology,pharmacy,and medicine in promoting a lab-toclinic translation of ICG based nano-medicine modalities.

Scheme 1.Schematic illustration of two kinds of tactics and mechanisms for indocyanine green based nano-drugs (nano-ICG) synthesis.(A) Verities of nanocarriers as“bullet shell”for ICG molecules loading and encapsulation.(B) ICG molecules itself will be a skeletal component of nano-ICG via self-assembling with various functional moieties that have low immunogenicity.

2.Customized nanocarriers for ICG delivery

With the rapid development of biotechnology and nanotechnology,customized nanocarriers come in diverse forms based on their desired capabilities and characterizations [23].For instance,we can choose the corresponding nanocarriers for improving solubility and bioavailability,altering bio-distribution,facilitating entry into target cells,and preventing the occurrence of severe side effects of the target molecules.Generally speaking,the cargo-carriers are nanomaterials that have a desired cavity around which to wrap the cargo,just like a“bullet shell”[19].These kinds of cargocarriers come in diverse forms (e.g.,mesoporous materials,metal organic fractures,polymeric nanocarriers and lipid-based nanocarriers).Though the different material sources,their drug loading principles are alike between each other.In this section,we will select two representative examples,polymeric nanocarriers and lipid-based nanocarriers,to introduce their principles of ICG loading and their further applications.

2.1.Polymeric nanocarriers

Polymeric nanocarriers are derived from a wealth of sources including hybrid nanoparticles,dendrimers,nanogels,nanocapsules,polymeric micelles,etc.Due to their similar cavity structure of these nanocarriers,the ICG molecules will be directly encapsulated into or via covalent linkage loading within [24,25].The most fundamental and significant merits of them are benefiting for enhancing ICG solubility,loading capacity and acting in synergy with other functional moieties and the most commonly used polymer molecules have been selected as representative for their detailed mechanism interpretation.

Hyaluronic acid(HA)is naturally existing,linear polysaccharide and distribute through the body as an essential constitute which is made up of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine[26].A wide assortment of polymeric materials can be synthesized through conjugation of hydrophobic moieties to the hydrophilic HA polymer backbone.Together with the inherent targeting ability for the overexpressed CD44 in cancer cells,it is an ideal carrier for functional moieties delivering to tumors [27].Recently,Cai et al.[28] fabricated one kind of metal-based polymeric nanoparticle via the self-assembled Febased metal-organic framework (MOF) and hyaluronic acid (HA) coupling as well as ICG loading (Fig.1).The excellent capability for ICG performance in terms of FLI and MRI intensity,desired corresponding tumor targeting ability,and photothermal treatment effects,has been demonstrated via in vivo and in vitro experiments.The same principle and ICG loading mechanism were used for iron oxide nanoparticles,Wang et al.[26] successfully decorated the iron oxide nanoparticles with the ICG and HA via electrostatic interaction for hybrid material synthesized.Benefiting from the superior capability of each component,this kind of composite could realize effective PA/PTT/FL imaging-guided cancer theranostics.More importantly,the metal-based polymeric nanoparticle can generate fetal ROS via intracellular Fenton reaction for cancer cell killing which promote synergistic therapeutic effects [26].

Generally,the raw materials of polymers come directly from nature such as poly D,L-lactic-co-glycolic acid (PLGA),polyethylene glycol(PEG),poly ε-caprolactone (PCL),and other multi-polymers.Among these materials,PLGA is another FDA-approved drug delivery system featuring good biodegradable ability in normal physiological conditions.Based on these merits,Chen et al.[29] cleverly prepared one kind of polymer with PLGA and ICG as well as imiquimod (R837) which act as an encapsulating polymer for phototherapeutic agent and immune response activator respectively.Through the oil-in-water (O/W) emulsion method,PLGA can encapsulate the other two molecules for synthetic polymers(PLGA-ICG-R837)(Fig.2).In a rigorous experiment procedure,this kind of polymer exhibited synergistic treatment with PTT and immunotherapy for more effective cancer treatment.Among these components,ICG molecules were used for PTT ablation of primary tumors and for generating tumor-associated antigens.Afterward,the released antigens,in combination with the R837-containing nanoparticle adjuvant,would generate strong immunological responses.Compared to the free ICG,not only does this kind of synthetic polymer improve the stability and distribution of encapsulating ICG in vivo,but,consequently,immunological functions were endowed for cancer immunotherapy.Apart from PLGA polymers,other multi-polymers such as PEG and PCL,have been used for ICG loading for further applications [30–33].

In addition to these universal polymers mentioned above for a variety of moiety packaging,some positively charged polymers may be superior for ICG delivery because of the inherent negatively charge of ICG.The cationic polymers have an inherent ability to prevent the leakage of loading drugs mainly due to the electrostatic interactions within the carriers and molecules.For example,cationic polyetherimide(PEI) and polydopamine (PDA) can load ICG by electrostatic interactions to hydrophobic electrostatic hybrids [33–35].Further binding of specific targeting ligands (e.g.,folic acid (FA),RGD) and other therapeutic drugs (e.g.,doxorubicin (DOX),tirapazamine (TPZ)),these peculiar polymers prominently bestow the ICG with a targeting ability and a theranostics capability [36,37].

2.2.Lipid-based nanocarriers

Fig.1.ICG encapsulation within polymeric nanocarriers to improve its capability.(A)Schematic representation of the synthesis procedure of HA together with MOF based nano-ICG.(B) TEM image of this kind of nano-ICG.(C) Absorbance spectra of different components.(D) In vivo PA images and T2-weighted MR images with nano-ICG treatment[28].Copyright:2016,American Chemical Society.(E)The synthetic route of iron oxide nanoparticle-based nano-ICG with HA coating and their schematic illustration of their biological functions in cancer theranostics.(F) SEM and TEM images of the synthetic nano-ICG [26].Copyright:2019,Wiley-VCH Verlag GmbH &Co.KGaA.

Fig.2.The PLGA based nano-ICG formulation and their great performance for cancer eradication.(A)Schematic illustration of synthetic procedure.(B)Hydrodynamic diameters of PLGA based nano-ICG measure by DLS,TEM image and its UV–vis–NIR spectra compared with free ICG.(C) The mechanism of cascaded anti-tumour immune responses induced by PLGA based nano-ICG with PTT in combination with checkpoint-blockade [29].Copyright:2016,Springer Nature.

The cytomembrane structure is the most important sharing component for mammalian cells,bacteria,and viruses[38].Benefiting from sharing the phospholipid bilayer,the lipid nanoparticles have become one kind of popular nano-formulation with regard to biocompatibility and biosecurity[39].What's more,as one kind of flexible nanomaterial,just like the cell membrane,the distinctive size-dependent properties and membrane fusion peculiarity could guarantee efficient cargo accumulation in tumor regions and delivery to the targeted cells [40].Herein one important example is that DOX loading liposomes have been approved by the FDA for tumor destruction.Therefore,this kind of specific liposome will be an excellent carrier for ICG delivery with the biosecurity guaranteed.

Generally,based on the amphiphilic peculiarity of liposomes,both water-soluble drugs and water-insoluble drugs can be loaded into the inner aqueous compartment or hydrophobic compartment of the phospholipid bilayer.Recently,Dai et al.[5] constructed a thermalsensitive liposome nanoparticle.These kinds of specific hybrid nanoparticles were constructed via ICG,DOX together with gadolinium(Gd)co-encapsulating into the liposome shell (Fig.3).Profiting from the great fluorescence property of ICG,the excellent chemotherapeutic effects of DOX,and the desired MRI contrast agent of Gd,these kinds of synthetic nanoparticles can integrate a FL/PAI/MRI triple-modal imaging strategy and chemo/PTT/PDT methods into one architecture.In the end,this specific hybrid structure successfully achieving multimodal imaging-guided cancer combination therapy and providing a promising platform for tumor tracing and eradication.Apart from these mono-disperse functional molecule polymers,other macromolecular polymers will be encapsulated together to improve their performance.For instance,the polymer PLGA could be entrapped with ICG before being packaged into the liposome to overcome the premature release before arriving ate the targeted site [41].

Given that the most vital characteristics for the desired nanomaterial are biosecurity and lower immunogenicity,the biological materials have obvious advantages over chemically synthesized ones.As an alternative to chemically synthesized liposomes,recently-developed cell membrane vesicles (MVs) have been widely applied to many kinds of disease treatment and curative effect affirmations [42,43].For instance,Ye et al.[44] chose red blood cell (RBC) membranes and the hydrophobic chemotherapeutic agent 10-Hydroxycamptothecin(HCPT)to construct camouflaged nano-ICG(RBCs@ICG-HCT).Small molecular drug assemblies of ICG and HCPT were enveloped into an external RBC shell,which dramatically improving the accumulation at the tumor sites via passive targeting and enhancing stability.Furthermore,with the cRGD peptide anchor on the surface,their active targeting effi-ciency and great membrane fusion capability will be improved significantly (Fig.4) [45].

Although the MVs directly constructed from varieties of functional cells have the greatest performance (e.g.,RBCs,macrophages,cancer cells),the major breakthrough of the “hypothesis of signal sequence”gives us the ability to genetically engineer cell membrane with artificial proteins.The component of cell surface proteins will be customized via this method and following prepared MVs will be endowed desired functions[46].For example,Zhang et al.[47]genetically displayed the human epidermal growth factor (hEGF) and anti-HER2 affibody as tumor targeting ligands on the HEK293T cell lines (Fig.5).After the loading of the ICG into the extracted MVs from these engineered cell lines,the biological compatibility and stability were greatly improved.Benefiting from the targeting moieties linkage,the targeting ability of ICG to the MDA-MB-468 xenograft model has improved compared with the control one.Apart from this simple amino acid sequence engineering,Liu et al.[48] selected the specific monoclonal antibody(anti-glypican 3 (GPC3) antibody (hGC33)) as the targeting ligand for tumor targeting.After the full-length hGC33 specifically expressed on the HEK 293T cell membrane surface,the targeting ability of prepared MVs was greatly enhanced.Their excellent tumor targeting performance and adjuvant therapeutics effect have demonstrated via rigorous experimental results in vitro and in vivo.

The aforementioned carrier materials successfully encapsulating ICG have several advantages.Firstly,with regard of physicochemical property of these nanomaterials,the encapsulation of ICG can effectively extend the circulation time.Then,from the perspective of optical imaging,the entrapped ICG displays a greater optical imaging effect depending on the specific accumulation and controllable release at the targeting sites.Finally,under the guidance of precise imaging,surgeons can perform resection more accurately,or provide a personalized therapeutic schedule.

Fig.3.Liposome based hybrid nano-ICG synthetic procedure and their tumor therapeutic characteristics.(A) The phase transition of the temperaturesensitive liposome was triggered by NIR-induced hyperthermia.(B) Characterization of this nanostructure (DLS and TEM image).(C) Photoacoustic imaging of tumors in vivo at the indicated time points after intravenous injection with synthetic nano-ICG[5].Copyright:2019,American Chemical Society.

Fig.4.Schematic illustration of the design of light activatable nano-ICG by a process involving RBC membrane cloaking strategy together with ICG and cis-platinum(II) co-packaging.(A) RBC membrane-based nano-ICG synthetic procedure.(B) Physicochemical characterizations of this kind of nano-ICG (TEM image).(C) T2-weighted MR images of tumor bearing mice after different treatments [45].Copyright:2018,Wiley-VCH Verlag GmbH &Co.KGaA.

Fig.5.Genetically engineered liposome-like nanovesicles as active targeting carriers for ICG delivery.(A)Schematic illustration of genetically engineered membrane vesicle extraction from the cell lines with the help of surfactants.(B)TEM images of prepared nanovesicles.(C)Absorption spectra of nanovesicles after ICG loading into inner cavity.(D)In vivo FL imaging of tumor bearing mice after i.v.injection of ICG loading nanovesicles at different time points[47].Copyright:2017,Wiley-VCH Verlag GmbH &Co.KGaA.

3.ICG-based self-assembly

With the central dogma of delivering the functional molecules to the target lesions,the nanocarriers may provide possible solutions because of their innate ability,such as the EPR effect,as mentioned above.In the process,the major issue for clinical application is the concern regarding biosafety during the synthesized nanocarrier degradation,metabolism,and excretion in vivo.Considering the characteristics of ICG such as sulfonic acid groups and polycyclic structure,ICG molecule can assemble with a variety of structural or functional ingredients through electrostatic interaction,hydrophobic effects,π-π stacking effect.In other words,the ICG itself will consider the functional moieties and skeletal component of synthetic nano-ICG.The main point to make here is that the jointly assembled materials—such as proteins,peptides,nucleic acid,and metal ions—are nontoxic [49,50].In this section,a developed innovative portfolio strategy for an ICG-based self-assembly system will be highlighted.

3.1.Proteins-based self-assembly

The concept of molecular self-assembly is inspired by ubiquitous natural processes such as protein folding,the formation of protein aggregates,and the pairing of nucleotides [51].It is a phenomenon that spontaneously forms stable,structurally ordered aggregates under noncovalent bonds.The forces mediate the self-assembly notably including ionic bonds (electrostatic interactions),hydrogen bonds,hydrophobic interactions,water-mediated hydrogen bonds,π-π stacking,and van der Waals interactions[49].Therefore,the formation of protein aggregates,and pairing of nucleotides,biological macromolecules may be an outstanding choice for ICG molecule assembly.For instance,FDA approved human serum albumin (HSA) has been widely applied to nanocarriers for loading hydrophobic,aromatic and charged compounds via intravenous administration and has the potential to be developed for coassembly complexes with considerable molecular drugs.Chen et al.[52] successfully fabricated one kind of HSA-based nano-ICG in which HSA,Paclitaxel (PTX),and ICG were hijacking for self-assembly(Fig.6).Utilizing this kind of HSA-based nano-ICG,highly effectively combined PTT and chemotherapy under the guidance of NIR imaging conducted,featuring the great solubility and prolonged blood circulation time.Apart from the HSA,other functional proteins such as the bovine serum albumin(BSA),ovalbum(OVA)and C-phycocyanin(CPC)have been widely used to prepare protein-based nanocarriers for ICG molecules because of abundant active groups for ICG conjugating[53,54].Besides,with the additional functions of these proteins,such as CPC can act as a tumor-associated macrophages targeted vehicle and OVA is a model antigen,the fabricated nano-ICG will significantly improve their accumulation in the tumor and photothermal-immunotherapy efficiency for precise and efficient cancer theranostics.

3.2.Peptides-based self-assembly

Fig.6.HSA based nano-ICG synthesis and application.(A) Schematic illustration of the formation of HSA based nano-ICG.HSA,PTX and ICG will spontaneous assembly in the aqueous solution to form nanoparticles.(B) TEM images of this kind of nano-ICG.Inserts:photos of the samples in PBS [52].Copyright:2014,Wiley-VCH Verlag GmbH&Co.KGaA.

Despite the great capacity for different kinds of functional proteins for protein-assembly-based nano-ICG,the inherent cross-reactive immunogenicity from different species is a potential risk factor for consideration[55].Thus,the peptides that act as the alternatives for these proteins have shown excellent performance in antigen delivery and targeting ability and as a blocking reagent for immunotherapy [56].In recent years,peptide-based self-assembly has been developed for the fabrication of various functional nanomaterials thanks to its inherent properties,such as good biocompatibility,tunable morphology,easy availability,controlled degradation,and chemical versatility [57].In addition,various peptide assemblies can be tailored to exhibit different active functions through adjustment of the amino acid sequence [58].For example,Huang et al.[59] co-assembled ICG and enzyme-responsive peptide precursor into a nanofiber after digestion by alkaline phosphates (ALP) likely by a head-to-tail arrangement (J-aggregates)(Fig.7).It was demonstrated that this kind of enzyme-initiated reaction can specifically occur in tumor region.When ICG self-assembled into Jaggregates,obvious red shift can be observed from 780 to 808 nm as well as narrower and stronger absorption peak.The fluorescence intensity was partially quenched and the reduced radiant energy mainly contributes to PA imaging and PTT properties.The head-to-tail crosslinked nanofibers might make π-electrons delocalized and cause electrons transition to a lower energy level.This design ingeniously optimized the physical and chemical properties of ICG,and at the same time made it play a good role in vivo arriving at admirable tumor sites and avoiding undesirable uptake and clearance.Apart from this kind of ALP-based responsive peptides,other functional peptides such as glutathione (GSH) can promote a novel cascaded multi-responsive19F probe (1-ICG nanoparticles) synthesis [60].This specific nano-ICG architecture can be used for accurate sensing and imaging of tumors through stepwise two stage19F signal activation/amplification.

3.3.Metal ions-based self-assembly

As mentioned before,one of the main influence factors for spontaneously molecular self-assembly is the binding force among the various components.The strength of the binding ability directly affects the physicochemical properties of nano-ICG and an exploration of the desired mediators for strong binding ability is needed.As we know,there is a substantial number of metalloproteins consisting of both metal ions and cofactors via coordination interactions in our organisms;hemoglobin is the representative [61].Some smart metal-nanodrug was constructed,inspired by the multicomponent self-organization of peptides,pigments,and metal ions in metalloproteins [20].The common coordination metal ions are zinc ions(Zn2+),manganese ions(Mn2+),iron ions (Fe3+),platinum ion (Pt2+) and other trace metals which play great roles in maintain the homeostasis [62–66].The biggest advantage of metallo-nanodrugs is the facile synthetic method due to the strong metal coordination.It is exciting to note that they possess robust colloidal stability in a normal physiological environment and present burst release in tumor microenvironments[67].Besides,these metal ions can provide coordination sites for molecules linkage together with their own properties for contrast agents.For instance,the Mn2+and Fe3+are commonly used MRI agents.When combined with ICG,the formed nano-ICG could integrate the strengths of ICG and metal ions.Shi et al.[64]designed a kind of ICG-metal ion hybrid nanoparticle that was assembled by Mn2+and ICG molecule under the protection of poly (vinylpyrrolidone) (PVP).The utilizing of PVP contributed to the coordination effect between Mn2+and ICG,and the final synthetic nano-ICG had more stable optical property and slower body clearance than the free one.Benefiting from their own imaging capacity,this kind of synthesized nano-ICG successfully integrated FL,PA and MR three-mode imaging and PTT treatment.In another case,Lin et al.[65]also constructed a Fe3+mediated ICG super-molecular assembly,which was synthesized through crosslinking between anionic sulfonic groups in ICG and Fe3+ions(Fig.8).The pre-synthetic Fe3+/ICG nanoclusters were further encapsulated into a microbubbles system to realize sonodynamic therapy against orthotopic hepatocellular carcinoma (HCC).Under the protection of microbubbles,the limitations and instability of ICG were almost overcome.Intriguingly,far more ROS were produced than free ICG after ultrasound,resulting in an effective optical and acoustic signal for a great imaging-guided sonodynamic anti-tumor effect.

3.4.Small molecules-based self-assembly

Fig.7.Tumor-specific phosphatase-triggered co-assembly strategy to form ICG doped nanofibers for cancer theranostics.(A) Schematic conversion of micelles to nanofiber via an enzyme-catalyzed dephosphorylation and their corresponding physical characteristics(TEM images and AFM images).(B)TEM images of nanofibers at different aging time points with ALP-catalyzed process.(C)Scheme of in situ conversion of micelles to nanofiber after intravenous injection.(D)Representative PA images HeLa tumor tissues after intravenous injection [59].Copyright © 2015 American Chemical Society.

Fig.8.Fe3+mediated ionic cross linking self-assembly for nano-ICG synthesis and their further applications for cancer sonotheranostics.(A)Schematic illustration of the fabrication of nano-ICG with Fe3+,ICG together with microbubble components of cholesterol and lectithin.(B)Antitumor evaluation and multi-model imaging of nano-ICG in orthotopic mouse models of HCC.(C)Time-dependent whole-body FL images of orthotopic HCC-bearing mice treated with synthetic nano-ICG and sonoactivation [65].Copyright:2019,The Royal Society of Chemistry.

Fig.9.Chemotherapeutic cargo and photothermal agent co-self-assembling nano-ICG for fluorescence and photoacoustic dual-modal imaging-guided synergistic therapy.(A)Preparation procedure of EPI nano-ICG:EPI and ICG can self-assembly via electrostatic,π–π stacking and hydrophobic interactions followed by surface PEGylation.(B) TEM images of synthetic EPI nanoICG (without PEGylation).(C) In vivo FL imaging and PA imaging of tumor bearing mice at different time point with the free ICG and EPI nano-ICG treatment [71].Copyright:2017,Elsevier B.V.

Other than the functional proteins,peptides or the trace metal ions which are essential for human body have inherent negligible biotoxicity or immunogenicity,recently developed molecules self-assembly directly has drawn increasing attentions either.This smart strategy provides a facile and green carrier-free drug delivery system which will reduce the potential toxicity of other ingredients but significantly increasing blood circulation time and overcoming the problem of low agents loading [68,69].Recently,Li et al.[70] successfully take advantages of epirubicin (EPI),DOX and ursolic acid (UA) of co-assembling capacity with photosensitizers for nano-ICG preparation.The molecular structure of EPI has anthraquinone rings,amino sugar and adjacent hydroxyl groups imparting itself hydrophobic and hydrophilic characters respectively.Then together with the ICG and DOX co-assembly,one specific carrier-free nano-ICG was synthesized (Fig.9)[71].This nanodrug revealed deeper penetration in tumor tissues and realized complete elimination of tumors even without recurrence under the cooperation of PTT and chemotherapy.In order to further control cytotoxic drugs release,DOX could be optimized via conjugating with responsive chemical bonds such as disulfide bond (s-s) [72].At high GSH concentrations in tumor regions,chemotherapeutic drug DOX gradually released avoiding early leak before reaching the target.They all significantly enhanced both chemotherapeutic drugs and ICG loading rates.In the end,this novel self-delivery without carrier strategy gets rid of defects of traditional nanocarriers encapsulating suffering from drug loading rate,systematic safety,multi-component complexity and batch difference [73,74].In addition to simple making process,this kind of drug assembly strategy can act as an efficient platform for synergistic chemotherapy dispose of traditional therapies facing.Together with the other kinds of functional moieties linkage such as targeting peptides or other ligands,this kind of nano-drug will be an ideal choice for cancer synergistic treatment.

4.Conclusions and perspectives

In summary,this review emphasizes two kinds of nano-ICG through encapsulation into nanocarriers or self-assembly methods with enhanced properties for cancer tracing and eradication (Table 1).Both of methods have their own advantages.It is obvious that nano-ICG can improve the free ICG solubility,enhance the loading rate,prevent the ICG from leaking and removal in vivo.When the carriers further decorated with other functional ingredients,such as targeting peptides/ligands and responsive motifs,nano-ICG can be further improved with the active targeting capability,controllable release efficiency,and optimized physicochemical properties,respectively.Notably,nano-ICG synthesized via self-assembly can be prepared by simply mixing and stirring but without considering the exogenous substances.Usually,the co-assembly motifs are peptides,proteins,metal ions existence in the body and some molecular drugs that almost have little toxicity.Besides,this kind of nano-ICG presents great biocompatibility and synergetic effect because their great ability for other functional ligands mixed.Despite the multitudinous advantages mentioned above,some intrinsic and potential risks are still unsolved for synthetic nano-ICG and willsignificantly limiting their further applications.For example,the vast majority of chemically synthesized materials have potential immunotoxicity and smart tactics for these materials decoration are need.Self-assembly nano-ICG is notable for its simple and green synthesis and great biocompatibility,but needs to improve the pluralistic functions.Moreover,it seems that single strategy could not make up for all deficiencies.If we take the positives and discard the negatives of both strategies by preparing self-assembled active cores and entrapping ICG with biosafety nanocarrier shells,nano-ICG could one day successfully move from bench to bed.

Table 1 Verities of nano-ICG synthetized via ICG nanocarriers encapsulation or self-assembly with other functional ligands.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Major State Basic Research Development Program of China (2017YFA0205201),the National Natural Science Foundation of China (NSFC) (81801817,81901876,and U1705281),the Fundamental Research Funds for the Central Universities (20720190088 and 20720200019),Fujian Undergraduate Training Program for Innovation and Entrepreneurship (201812631010,JT180649,and HXJB-08),Education and Scientific Research Project for Young Teachers of Fujian Province (JAT190853),and the Program for New Century Excellent Talents in University,China (NCET-13-0502).

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pnsc.2020.08.002.