Black phosphorus : A novel nanoplatform with potential in the ̄ eld of bio-photonic nanomedicine

Singleor few-layer black phosphorus (FLBP) has attracted great attentions in scienti ̄c community with its excellent properties, including biodegradability, unique puckered lattice con ̄guration, attractive electrical properties and direct and tunable band gap. In recent years, FLBP has been widely studied in bio-photonic ̄elds such as photothermal and photodynamic therapy, drug delivery, bioimaging and biosensor, showing attractive clinical potential. Because of the marked advantages of FLBP nanomaterials in bio-photonic ̄elds, this review article reviews the latest advances of biomaterials based on FLBP in biomedical applications, ranging from biocompatibility, medical diagnosis to treatment.

2][123][124] As the result of the direct and tunable bandgap, few-layer black phosphorus (FLBP) processes a widely light absorption indicating a great potential in photothermal and photodynamic therapy.2][133][134][135][136][137] Due to the balance of these features, BP is often occurred in biosensing and bioimaging ¯eld.FLBP, with the unique puckered lattice con¯guration, possesses much larger surface area-to-volume ratio than other 2D materials, which results in extremely high drug loading capacity. 138Furthermore, FLBP displays negligible toxicity and outstanding biodegradability in the biological environment which distinguishes it from other common 2D materials.FLBP is completely degradable in vivo, and the products, such as phosphite ions, phosphate ions and other P x O y , are nontoxic and can be easily exhibited a desire renal ¯ltration.In addition, FLBP possesses attractive electrical characteristics, unique band structure and natural biocompatibility, indicating that FLBP has a great potential for biomedical ¯eld. 37P has aroused more and more research interest on its biomedicine applications with its unique physical and chemical properties.However, there is still a lot of problems needs to be solved to achieve the requirements of clinical applications, such as stability, [139][140][141][142][143][144] in vivo toxicity, 145 biodegradation, 146 excretion, 147 etc.In order to meet the urgent demand for the novel 2D materials used in biomedical ¯eld, it is necessary to summarize the latest achievement of BP in bio-photonic ¯elds.In this paper, we mainly review the biomedical research of BP in biological diagnosis and therapy.

Biocompatibility of BP
Lati® et al. 145 established human lung carcinoma cancer epithelial cells (A549) model to study the toxicity of FLBP.To ensure the credibility of the results, graphene and MoS 2 are used as comparison and two similar methods based on similar principles were used to assess cell viabilities.In this work, the toxicity of FLBP increases with increasing concentration, when the concentration of FLBP is below 50 ppm.They found that toxicity of FLBP is lower than that of graphene but higher than that of MoS 2 .Due to the limitations of the test method and the size of FLBP used in the paper, there are still a lot of toxicity assessments to do before the clinical applications of FLBP.Zhang et al. 148 studied the dependence of FLBP toxicity on size, concentration, exposure time and cell type, as presented in Fig. 1.FLBP was fabricated in oxygen-free Millipore water, and centrifugation at di®erent speeds was used to obtain FLBP with three di®erent sizes, named BP-1, BP-2 and BP-3, from large to small.The results of this paper indicate that FLBP with the BP-3 dimensions is appropriate for biomedical applications.They proposed and veri¯ed two possible mechanisms of the cytotoxicity of FLBP: (1) FLBP produces reactive oxygen species (ROS) to kill cells and (2) FLBP destroys cell membrane integrity.The cytotoxicity of BP-1 is much higher than that of BP-3.Fortunately, the size of BP-3 happens to be the most widely used size of FLBP nanosheets in the biomedical ¯eld.The results of this paper indicate that FLBP is appropriate for biomedical applications.Mu et al. 149 explored toxicological studies on BP quantum dots (BPQDs), which underwent faster renal ¯ltration than BP nanosheets, systemically with cell and animal model.Comparing the cells viability and the endocellular ROS levels in experimental group with different concentrations of BPQDs and the control group, they found that the cytotoxicity of BPQDs is mainly derived from ROS produced by itself.The results of in vivo assays indicated that catalase activity in liver will reduce 24 h after injection, but there was no obvious adverse e®ect after a week without recurrence after a month.Song et al. 150 studied the dependence of FLBP cytotoxicity on dose and time.When the concentration exceeded 4 ppm, FLBP showed signi¯cant cytotoxicity, which con°icts with other works probably for the reason of the di®erence in the size of FLBP.
The percentage of live cells was detected within 12 h at a dose of 10 ppm and was signi¯cantly reduced after 6 h, indicating the need for e®ective modi¯cation before BP achieves clinical applications.

Medical Diagnosis 3.1. Biomolecular biosensors
[153][154][155][156][157][158][159][160] Chen et al. 161 investigated FLBP for human immunoglobulin G (IgG) detection.FLBP was fabricated by a mechanical exfoliation method and passivated with Al 2 O 3 .The FLBP-based device showed rapid response performances and excellent sensitivity ($10 ng mL À1 ) to human IgG.Furthermore, the FLBP-based device exhibited good stability without obvious changes in performance.Mayorga-Martinez et al. 162 obtained FLBP with the size of 40 $ 200 nm by electrochemical exfoliation.The as-fabricated FLBP showed active electrocatalytic performances for the hydrogen evolution reaction and IgG detection.In addition, FLBP with poly-L-lysine (PLL) displays a great potential in label-free detection of myoglobin (Mb), which is an important signal of cardiovascular events. 163FLBP was created by liquid-phase exfoliation (LPE) with an aqueous surfactant solution in argon atmosphere, and PLL was used to functionalize FLBP in order to accelerate binding with anti-Mb DNA aptamers.The PLL-BP dispersed stability in aqueous medium and showed a stable detection performance in phosphate-bu®ered saline (PBS) and serum samples, indicating successful surface modi¯cation.The FLBP-based device possesses a high sensitivity (36 A pg À1 mL cm À2 ) and a low detection limit (0.524 pg mL À1 ), with a widely dynamic response range (1 pg mL À1 to 16 g mL À1 ).
In addition to excellent electrical properties, the unique °uorescence properties of FLBP were also applied to biological detection.Gu et al. 164 investigated BPQDs with a sonication-assisted solvothermal method for the applications of acetylcholinesterase activity sensing probes.The BPQDs showed strong green °uorescence at 497 nm and an extremely high quantum yield (8.4%).Furthermore, pH relevant °uorescence property with reliable photostability had been observed in this work.Lee et al. 165 prepared BPQDs with a strong visible blue-emitting performance by LPE in various organic solvents.They used catechol-grafted poly(ethylene glycol) (CA-PEG) to functionalize BPQDs in basic bu®er to achieve a stable dispersible in water and an extremely low cytotoxicity.The photoluminescence emission centered of PEG-BPQDs is 428 nm and the photoluminescence quantum yield is %5% when the excitation wavelength is 365 nm.Yew et al. 166 demonstrated FLBP as a platform in °uorescence-based DNA biosensors.BP crystals transformed from red phosphorus allotrope in the high pressure, and FLBP was synthesized by share force milling at 17,000 rpm.FLBP showed an obvious photoluminescence emission centered at 527 nm with an excitation wavelength of 200 nm, indicating potential applications as °uorescent sensing platform.This biosensor shows a low detection limit (5.9 pM) and quanti¯cation limit (19.7 pM).In addition, an excellent linearity (r ¼ 0:91) and a widely dynamic response range (4-4000 pM) were observed in this work.

Tumor imaging
8][169][170][171][172][173][174][175][176][177] With the enhanced permeability and retention (EPR) e®ect, [178][179][180][181][182][183][184][185][186][187][188] FLBP can be passively enriched to the tumor site, so the tumor imaging based on FLBP has received extensive attention.Shao et al. 147 investigated that BPQDs, which manufactured by the simple liquid exfoliation method, with poly lactic-co-glycolic acid (PLGA) were used to modify BPQDs in order to enhance the stability and solubility of BPQDs in water.As-fabricated BP-PLGA can e®ectively passively accumulate to the tumor region with tail vein injection because of the EPR e®ect.Figure 2(a) demonstrates the infrared thermographic images of the mice irradiated by near-infrared (NIR) laser 24 h after injection.Drawing a comparison between the tumor temperature of the test groups and of the control groups under the same power irradiation, it is obvious that the tumor temperature of the test groups (26.3 C of BP-PLGA) increased much more than that of the control groups (6.2 C, 7.8 C and 10.8 C of PBS, PLGA and bare BPQDs, respectively), indicating the excellent photothermal performance of BP-PLGA.In addition, many other researchers also have achieved extremely good photothermal imaging by di®erent modi¯cations of FLBP. 189,190un et al. 191 investigated BPQDs with excellent photostability for the applications of photoacoustic (PA) imaging, as shown in Fig. 2(b).The PEGylated BPQDs with a uniform size were fabricated by a simple high energy mechanical milling method.When the concentration of the PEGylated BPQDs is in the range of 0-250 g mL À1 , the intensity of the PA signal rises linearly as the concentration increases.Furthermore, the PA signal intensity in tumor was still higher than that of liver and kidney, 24 h after injection, representing a long retention time and EPR e®ect.Sun et al. 192 reported BPQDs loaded with titanium ligand (TiL4) as PA imaging agent.Dispersibility and stability of TiL 4 @BPQDs in water are much better than those of bare BPQDs.With increasing wavelength of the irradiation, the intensity of the PA signal decreased because the optical absorption of wavelength range from 680 nm to 808 nm reduces.
Yang et al. 193 prepared FLBP coated with Au nanoparticles for surface-enhanced Raman scattering (SERS) imaging.BP-Au NSs were fabricated with a facile re°ux method, and mPEG-SH was added to improve the dispersion and stability in water.The molecular mechanism of photothermal therapy (PTT) was investigated by SERS analysis.
The SERS analysis illustrates that PTT damages the membrane microstructure in tumor, and some BP-Au NSs appear in the nucleus region.

Phototherapy
7][218][219][220][221][222] The principle of PTT is based on the heat energy generated by the photothermal agent under irradiation to achieve the therapeutic e®ect.PDT agent can generate ROS to kill cancer cells under laser irradiation.FLBP has broadband absorption characteristics because of its tunable direct band gap, indicating great potential for the applications of cancer phototherapy.6][227] Some researchers explored new applications for the thermal performance of BP, such as post-surgical treatment of cancer, 3D-printed sca®olds and neuroprotective nanomedicine.

PDT
FLBP for PDT was fabricated by LPE in an aqueous solvent. 230At a wavelength of 530 nm, the quantum yield of FLBP producing singlet oxygen is very high (0.91).In vitro and in vivo FLBP experiments showed good PDT e®ects, as shown in Fig. 4. BPQDs were synthesized by LPE in N-methyl pyrrolidone and coated with PEG to achieve enhanced stability in water.Guo et al. 231 used BP dispersion to incubate cancer cells under 670 nm laser irradiation and researched the dependence of cell survival rate on BPQD concentrations, illumination time and laser intensity.The ROS generated by FLBP was able to kill the cancer cells e±ciently at very low concentration (1.6 ppm) and at very weak laser power (160 mW cm À2 ).Furthermore, 65% of BPQDs was found excreted with urine within 8 h, probably for the reason of the ultra-small hydrodynamic size of BPQDs, indicating that BPQDs have a good biocompatibility.Chen et al. 232 found that the bleaching signal of BPQDs is built up rapidly (<2 ns) and lasts a long time (100 s).The triplet generation attributed to intersystem crossing was observed and may be the reason of the highly e±cient singlet oxygen generation of BPQDs.Tan et al. 233 achieved in situ disinfection with PDT based on FLBP.FLBP was modi¯ed with poly(4pyridonemethylstyrene) endoperoxide (PPMS-EPO).PPMS not only enhances the stability of FLBP, but also stores singlet oxygen.The PPMS-EPO/BPS showed good photodynamic performance even in the absence of light, indicating a good clinical potential.

Therapeutic agent delivery
Chemotherapy is an e®ective cancer treatment, 234,235 but the high toxicity, 236 low targeting 237,238 and drug resistance 239 limit its therapeutic e®ect.In order to solve the aforementioned problems, researchers load therapeutic agents onto drug delivery systems (DDSs) to enhance chemotherapeutic e®ect.With large surface area-to-volume radio, unique pleated structure and excellent light response characteristics, FLBP has great potential for DDSs.Tao et al. 240 ¯rst applied FLBP to DDSs, as shown in Fig. 5. PEG-FA/Cy7-functionalized FLBP exhibited good biocompatibility, obvious tumor targeting and strong °uorescence signal and was loaded with doxorubicin (DOX) via electrostatic adsorption.In addition, the endocytosis pathway of the FLBP nanoparticle had also been screened.Chen et al. 138 prepared a drug loading platform with a pH/photo response based on FLBP.
Their results showed that FLBP has a very high DOX loading (980%) due to its puckered lattice con¯guration, large interlay distance 0.524 nm and negative charge.They used the therapeutic system to achieve synergistic treatment of PTT/PDT/ chemotherapy and achieved good anti-tumor e®ects.Wang et al. 241 used one-pot method to prepare HSA-modi¯ed FLBP, which can e®ectively load paclitaxel by hydrophobic interactions.Qiu et al. 190 fabricated a BP@hydrogel-based DDS.The DDSs enable intelligent light-controlled drug release and degradation, and the degradation products are completely nontoxic and easily metabolized.Yin et al. 242 fabricated FLBP loaded with interfering RNA as the applications of gene delivery systems.Compared with the commercial delivery reagents, the BPQDs exhibited a higher transfection e±ciency and low toxicity.

Summary
This paper summarizes the latest progress in FLBP research from three aspects, including biocompatibility of BP, medical diagnosis and medical therapeutic based on BP.Various factors such as size, concentration and test cell line can a®ect the toxicity of FLBP, but overall, FLBP shows relatively low toxic at e®ective dosing dose.Due to its attractive electrical properties, °uorescence characteristics and large surface area-to-volume ratio, FLBP shows a great potential for biosensors and imaging.In terms of biological therapy, FLBP mainly achieves therapeutic e®ects through its excellent optical response characteristics and as a drug delivery platform.
Although studies on the biomedical applications of FLBP have made a lot of progress, there are still some problems that need to be solved before its clinical translation.First of all, because the size of the FLBP has a great impact on toxicity and treatment e®ects, it is very important to ¯nd a novel method to fabricate FLBP with uniform size and high production.Secondly, other treatments such as chemotherapy, immunotherapy, etc. could be combined with BP to achieve synergistic e®ect.Finally, the targeted treatments based on FLBP should be developed for speci¯c diseases to achieve smaller side e®ects and better therapeutic e®ects.This requires multidisciplinary researchers to work together.FLBP has great potential in biomedicine.To promote the clinical applications of FLBP, we summarized the progress of FLBP in biomedical ¯eld.

4. 1 Fig. 2 .
Fig. 2. (a) Infrared thermographic images in the nude mice bearing MCF7 breast tumor under NIR laser irradiation (808 nm, 1 W cm À2 ) after intravenous injection at 24 h with di®erent treatments.(b) PA maps of PEGylated BP nanoparticles dispersions with di®erent concentrations (¯rst column), and time-lapsed PA images of liver, kidney and tumor of female BALB/c mice after intravenous injection with PEGylated BP nanoparticles (200 mL, 2 mg mL À1 ).
therapeutic and biodegradable.In vitro experiments showed that PLGA-BPQDs killed the most of cancer cells at an extremely low concentration (10 ppm).What's more, the ¯nal degradation products of PLGA-BPQDs are carbon dioxide, water, phosphate and phosphonate, which normally exist both in vivo and in vitro and result in little side e®ects.Fu et al. 224 prepared three types of FLBP with average size of 394 AE 75 nm, 118 AE 22 nm and 4:5 AE 0:6 nm by LPE method, named as L-BP, M-BP and S-BP.The temperature of L-BP solution with a concentration of 25 ppm was able to rise by 24 C under NIR laser irradiation for 10 min, whereas temperatures of M-BP and S-BP solutions with the equal concentration could only rise by 21.8 C and 19.2 C, indicating that L-BP has the best photothermal performance.

Fig. 3 .
Fig. 3.The dependence of cell viability on concentration of BPQDs and cell lines.The irradiation time is 10 min and power density of 808 nm laser is 1.0 W cm À2 .(a) Fluorescence images of cancer cells incubated with BPQDs after irradiation of 808 nm laser.(b) C6 cells viability after treatments with di®erent concentrations of BPQDs.(c) MCF7 cells viability after treatments with di®erent concentrations of BPQDs.

Fig. 4 .
Fig. 4. In vivo PDT.(a) Tumor volume of di®erent time post-injections.(b) Tumor pictures of experimental group and control group after treatments.(c) PCNA and TUNEL analysis of tumor tissues.