dendrimer is one of the first dendrimers that have been extensively studied.Among them,PAMAM dendrimer and polylysine dendrimer do not have the traditional fluorescence emission groups,but they can still produce autofluorescence effect,and the fluorescence intensity can be controlled by changing the conditions,so they can be directly used for biological imaging^[30,31]。 Based on the inherent fluorescence characteristics of dendrimers,a variety of functionalized delivery vehicles,nanoprobes and biomarkers can be constructed for bioimaging^[30⇓~32]。

Al-Jamal et Al.Prepared amino-terminated polylysine dendrimers（Gly-Lys₃₁(NH₂)₃₂and Gly-Lys₆₃(NH₂)₆₄）of generation 5(G5)and generation 6(G6)with molecular weights of 4047 and 8149 Da,respectively,stepwise by solid-phase peptide synthesis on 4-toluenehydroamine(MBHA)resin with a loading capacity of 0.67 mmol/gm^[30]。 Although these two novel polylysine dendrimers lack fluorescent groups,they still have sufficient fluorescence intensity at low concentrations.Among them,Gly-Lys₆₃(NH₂)₆₄has higher fluorescence intensity than Gly-Lys₃₁(NH₂)₃₂at the same molar concentration.The transport and uptake of Gly-Lys₆₃(NH₂)₆₄in human colorectal adenocarcinoma Caco-2 cells can be monitored by confocal laser scanning microscopy based on the relatively high intrinsic autofluorescence intensity of Gly-Lys₆₃(NH₂)₆₄,and the whole process of Gly-Lys₆₃(NH₂)₆₄adsorption to the cell surface,then entering the cytoplasm,and reaching the nucleus within 35-45 min can be observed.The diffusion coefficient of Gly-Lys₆₃(NH₂)₆₄in the cytoplasm can be calculated by measuring the change of fluorescence intensity in the target region with time,which shows that the diffusion coefficient of Gly-Lys₆₃(NH₂)₆₄in the cytoplasm reaches（6.27±0.49）×10^-11cm²·s^-1,which is 1000 times lower than that of Gly-Lys₆₃(NH₂)₆₄in water.The polylysine dendrimer with inherent autofluorescence can be used as a nanoprobe for monitoring cell transport,and the mechanism and process of cell transport substances can be monitored without fluorescent group labeling,which will be helpful for further study of its diffusion in living cells 。

Tsai et al.Used amino-terminated PAMAM dendrimer with inherent autofluorescence AS a platform to combine with three antisense oligonucleotide(AS-ODN)sequences p75,NGF1 and NGF2 through electrostatic interaction to prepare fluorescent PAMAM dendrimer(F-PAMAM)with different nitrogen/phosphorus ratios(N/P)to inhibit p75 neurotrophic factor receptor(p75NTR)or nerve growth factor(NGF)in rat glioma C6 cells^[31]。 based on the inherent fluorescence properties of F-PAMAM,the cellular uptake behavior of the F-PAMAM/as-ODN complex can be analyzed by fluorescence microscopy and flow cytometry without additional fluorescence labeling.the fluorescence intensity of that F-PAMAM/AS-ODN(p75)complex measure by flow cytometry showed that C6 cell had a relatively high uptake efficiency of the F-PAMAM/AS-ODN(p75)complex,and the average fluorescence intensity of each F-PAMAM/AS-ODN complex At different N/P ratios showed that the uptake efficiency of the antisense oligonucleotide sequence NGF2 by C6 cells was relatively low even at an N/P ratio of 40.Although the fluorescence intensity and definition of C6 cells transfected with F-PAMAM/AS-ODN(p75)complex were not comparable to those of the traditional dye labeling system under the fluorescence microscope,the slight enhancement of fluorescence intensity indicated that the cellular uptake efficiency increased with the increase of N/P ratio from 10 to 20.at N/P=20,atomic force microscopy analysis confirmed that p75 and PAMAM dendrimers formed well-condensed spherical particles with a size of less than 200 nm(fig.2).Based on the inherent fluorescence properties of F-PAMAM,it can be used AS a bifunctionalized gene carrier and fluorescent probe not only to determine the optimal N/P ratio for the delivery of different antisense oligonucleotides,but also to analyze the cellular uptake efficiency of F-PAMAM/AS-ODN(p75,NGF1 and NGF2)complexes in C6 cells.the fluorescent dendrimer-Based antisense oligonucleotide delivery system is convenient for researchers to intuitively analyze and evaluate the uptake behavior of cells,and is expected to be used AS a gene carrier to simultaneously realize delivery,transfection and biological imaging。

Wang et al.Used G5 PAMAM dendrimer as a platform to prepare a fluorescent dendrimer(F-G5)without fluorescent labeling by capping amino acetaldehyde,and the Schiff base C=N bond formed produced a strong green fluorescence signal through n-π*transition^[32]。 F-G5-PEG was further PEGylated to form F-G5-PEG.Based on its strong green fluorescence,it was found that F-G5-PEG had the ability of real-time cell tracking in human melanoma SKMEL28 cells.with the extension of incubation time to 24 H,the green fluorescence signal in cells increased significantly,and F-G5-PEG was mainly aggregated in lysosomes After endocytosis.after 24 H of incubation,bright green particles of F-G5-PEG were found at the edge of the cells,which was caused by the exocytosis of F-G5-PEG by SKMEL28 cells.F-G5-PEG was used as a nanoplatform to encapsulate the anticancer drug doxorubicin(DOX)by solvent displacement method to construct F-G5-PEG/DOX,which could monitor the cellular uptake of SKMEL28 cells through its unique dendrimer autofluorescence.Compared With G5 PAMAM dendrimer,F-G5-PEG improved the delivery efficiency of DOX and accelerated the release of DOX in cells in a slightly acidic environment.the novel F-G5-PEG nanoplatform lays the foundation for fluorescence tracking and controlled delivery of anticancer drugs。

Base on that autofluorescence of the dendrimer,the constructed various functionalize dendrimers can monitor the uptake behavior at the cell level without additionally loading a fluorescent reagent,so that the effective delivery of drugs and gene can be realized,and the cell uptake efficiency can be evaluated through the inherent fluorescence characteristics of the dendrimer,so that the real-time biological FL imaging can be conveniently carried out.However,the fluorescence effect of autofluorescent functionalized dendrimer is easily affected by the generation number,concentration of dendrimer and the pH of the environment where the capping group is located,which easily leads to the change of fluorescence intensity,thus limiting the application of autofluorescent functionalized dendrimer-mediated FL imaging.Therefore,how to optimize the dendrimer structure to reduce the influence of many factors on its autofluorescence properties and optimize the imaging efficiency of FL is still an urgent research topic。

dendrimer loaded with fluorescent dye molecules is the most common and widely used functionalized fluorescent reagent,which is typically characterized by the appearance of fluorescent properties of functionalized dendrimer due to the intervention of fluorescent groups.It can be divided into three forms:fluorescent dye end group modification,fluorescent dye physically wrapped and fluorescent group embedded in the core of dendrimer^[33⇓~35]。 According to the different fluorescent dye molecules,the fluorescent dyes often modified on the surface of dendrimers include:FITC,Cy,indocyanine green(ICG),etc^[33,36,37]。 Fluorescent dyes that are often encapsulated in dendrimers include Pc^[27,34]。 Fluorescent dyes that are often embedded in the core of dendrimers include PDI,FITC,etc^[35,38]。 the dendrimer loaded with fluorescent dye molecules not only improves the water solubility and stability of the small molecular fluorescent dye,but also endows the dendrimer loaded with fluorescent dye molecules with specificity by taking the dendrimer as a medium through functional modification,and can realize specific FL imaging of cancer cells or tumors^[27,36,39]。

as a typical organic fluorescent molecule,FITC fluorescent dye has an aromatic ring As its chromophore and is often linked to a variety of compounds by isothiocyanate groups.the covalent bonding of FITC to dendrimer can not only improve the water solubility of FITC,but also slow down the fluorescence quenching rate to a certain extent^[39]。 Michlewska et al.Modified the surface of carbosilane dendrimer（G1-[NH₂]₄）with 2-pyridinecarboxaldehyde,and then chelated dichloro(methyl isopropyl benzene)ruthenium(II)on the G1-[NH₂]₄with 2-pyridinecarboxaldehyde as a bridge,and covalently bonded fluorescent dye molecule FITC to the surface of dendrimer to obtain ruthenium-terminated hybrid carbosilane dendrimer CRD13-FITC^[33]。 the novel metal-based dendrimer has a good cancer cell treatment effect due to the existence of the transition metal element ruthenium.Modification of this functionalized dendrimer by FITC did not affect cellular uptake of CRD13-FITC,and the presence of FITC conferred CRD13-FITC cancer and normal cell tracking ability.Flow cytometry and confocal scanning microscopy showed that the uptake of CRD13-FITC in human leukemia tumor HL-60 cells was higher than that in normal PBMC cells after 24 H incubation.the cytotoxicity of CRD13-FITC on HL-60 cells was higher than that on PBMC cells when the concentration of CRD13-FITC reached 10µmol/L.the FITC-modified ruthenium-based dendrimer has both FL imaging and anticancer effects,and can be used for real-time monitoring of cancer cells,providing a basis for subsequent in-depth study of the therapeutic effect of cancer cells.Similarly,Nagai et al.Also used FITC as an FL imaging reagent,covalently bonded FITC-modified matrix metalloproteinase-2(MMP-2)substrate peptide(MMP-pep)to the surface of carboxylated dendrimer(COOH-den),and then further combined tumor-homing peptide(tLyP-1)with MMP-pep/COOH-den by amidation reaction to prepare MMP-2-responsive fluorescent probe tLyP-1/MMP-pep/COOH-en for lymphocyte^[39]。 the uptake of tLyP-1/MMP-pep/COOH-den by different cells was observed by fluorescence microscopy Under the guidance of FITC FL imaging.it was found that the uptake of tLyP-1/MMP-pep/COOH-den was higher in human fibrosarcoma HT-1080 cells and mouse breast cancer 4T1 cells with high MMP-2 expression,so the two cells showed obvious fluorescence signals;However,no fluorescence signal was observed in mouse macrophage RAW264 with low MMP-2 expression.At the same time,It was found that HT-1080 cells and 4T1 cells treated with matrix metalloproteinase inhibitor(GM-6001)did not show green fluorescence signal after incubation with tLyP-1/MMP-pep/COOH-den,indicating the dependence of fluorescence signal on MMP-2.under the same conditions,the mean fluorescence intensity of tLyP-1/MMP-pep/COOH-den in HT-1080 and 4T1 cells was higher than that in HT-1080 and 4T1 cells treated with MMP-pep/COOH-den without tLyP-1 modification,and FITC-mediated FL imaging could verify that the targeting of tLyP-1 enhanced the uptake of tLyP-1/MMP-pep/COOH-den in HT-.According to MMP activity,MMP-2 secreted lymph node metastatic cancer cells can be monitored by FITC-modified tLyP-1/MMP-pep/COOH-den observation,and tLyP-1 can further enhance cancer cell uptake.This dipeptide-functionalized dendrimer can be used as a matrix metalloproteinase-responsive fluorescent probe to effectively detect lymph node metastatic cancer cells by FITC-mediated FL imaging。

As a common biomarker,Cy fluorescent dyes have the characteristics of high fluorescence quantum yield,high extinction coefficient and weak background when labeled and developed based on their symmetrical indole base chromophore,so Cy fluorescent dyes are often used for in vivo imaging of mice^[40]。 However,Cy dyes with multiple structures are prone to aggregation,and their fluorescence tracing in vivo is not specific,so combining them with functionalized dendrimers to construct FL imaging reagents can greatly improve the defects of Cy dyes and improve the effect of FL imaging in vivo^[36,41]。 Chen et al.First prepared zinc-doped iron oxide nanooctahedra(ZIONOs)by thermal decomposition,then covalently bonded with G5 PAMAM-NH₂dendrimer by ligand exchange with 2,3-dimercaptosuccinic acid(DMSA),and modified with methoxylated polyethylene glycol(mPEG)and fluorescent dye Cy5.5,and finally modified with nucleoprotein-targeting aptamer AS1411 on the surface of G5 PAMAM-NH₂dendrimer to prepare ZIPP(Cy5.5)-Apt nanocarrier^[36]。 Anticancer drug DOX and mouse monoclonal antibody/rabbit monoclonal antibody small interfering RNA(HSP70/HSP90 siRNAs)are wrapped into a ZIPP-Apt nano carrier by taking G5 PAMAM-NH₂dendrimer as a medium to prepare a breast cancer targeting nano diagnosis and treatment complex ZIPP-Apt:DOX/siHSPs(Figure 3A).Based on the affinity of AS1411-nucleoprotein,the cancer cell uptake and tumor-specific accumulation of ZIPP-Apt:DOX/siHSPs were significantly increased,and it was further confirmed that the simultaneous depletion of HSP70 and HSP90 could induce cancer cell apoptosis,thereby achieving tumor-sensitive magnetic hyperthermia and chemotherapy.ZIPP(Cy5.5)-Apt,as a dual-modality near-infrared(NIR)imaging and MR imaging agent targeting 4T1 tumor-bearing mice,can be used to monitor the tissue distribution of nanoparticles in vivo and verify its breast cancer targeting effect.The results of in vivo FL imaging test showed that whether ZIPP(Cy5.5)-Apt was modified by targeting aptamer AS1411 or ZIPP(Cy5.5)-CRO was modified by control C-rich oligonucleotide(CRO),After injection into tumor-bearing mice via tail vein for 8 H,the nanocomplexes preferentially accumulated in the tumor,resulting in a strong red fluorescence signal in the tumor,which may be due to the high permeability and long retention(EPR)effect,resulting in the non-selective uptake of nanocomplexes of similar size by the tumor.However,at 24 and 48 H,the ZIPP(Cy5.5)-Apt-treated tumor-bearing mice had obvious red fluorescence signals,while at the same time point,the ZIPP(Cy5.5)-CRO-treated tumor-bearing mice had significantly reduced tumor fluorescence signals,indicating that ZIPP-Apt remained in the tumor at 48 H,while ZIPP-CRO was rapidly eliminated from the body(Figure 3B).The tumor targeting accumulation effect of ZIPP(Cy5.5)-Apt was confirmed by the NIR FL imaging of tumors and organs in vitro 48 H after material injection,and the in vitro tumor fluorescence signal of ZIPP(Cy5.5)-Apt-treated tumor-bearing mice was significantly stronger than that of the non-targeting control group ZIPP[Cy5.5]-CRO-treated tumor-bearing mice(Figure 3C).The functional dendrimer nanocomposite system modified by Cy5.5 can realize sensitive magnetic hyperthermia and chemotherapy guided by FL/MR imaging targeting the NIR region of breast cancer through the mediation of Cy5.5,and realize the in vivo tracking of tumor-bearing mice of the nanosystem through the fluorescence imaging mediated by Cy5.5 。

Tsuchimochi et al.Grafted the third generation(G3)PAMAM dendrimer on the surface of silica nanoparticles to form PCSNs nanocarriers,and covalently bonded PCSN with ICG by using the amino groups on the surface of PAMAM dendrimer,and chelated technetium-99m（^99mTc）at the same time to form a dual-modality imaging probe for sentinel lymph node monitoring,which can accurately predict the time of cancer progression^[37]。 In vivo animal experiments showed that low concentration ICG-mediated NIR FL imaging achieved real-time clear fluorescence monitoring of cervical lymph nodes,and 19 lymph nodes were found by dissecting 6 rats,of which 6 lymph nodes showed fluorescence signals from weak to strong,indicating that some cancer cells had metastasized.The corresponding change in intensity of radioactivity mediated by^99mTc was similar to the change in intensity of fluorescence in cervical lymph nodes mediated by ICG.The novel ICG-functionalized dendrimer compound not only can be used for real-time NIR region FL imaging monitoring of sentinel lymph nodes,but also provides a novel diagnostic means for cancer cell metastasis,and the degree of cancer cell metastasis is judged by the intensity of fluorescence signals 。

in addition to modifying the end group of fluorescent dye molecules on the surface of dendrimers,fluorescent dyes can also be physically wrapped inside functionalized dendrimers to mediate tumor FL imaging,such as Pc dyes.Based on their excellent NIR optical properties,Pc fluorescent dyes can be used not only for FL imaging-guided drug delivery,but also for deep non-invasive treatment of tumors via photodynamic therapy(PDT).However,due to its poor water solubility,Pc is easy to aggregate in aqueous medium throughπ-πstacking and hydrophobic interaction,which leads to the self-quenching of its excited state,and it is not specific to cancer cells,resulting in its limited clinical application^[34]。 In order to solve this problem,Taratula et al.Encapsulated monosubstituted phthalocyanine(PcSi(OH)(mob))into the fourth generation of polypropylene imine(PPI)dendrimer,and then modified the surface of PPI dendrimer with PEG and luteinizing hormone-releasing hormone peptide(LHRH).In order to improve its biocompatibility and cancer cell selectivity,a Pc-loaded functionalized dendrimer nano-diagnostic reagent Pc-LHRH was developed for targeted delivery of Pc to LHRH receptor-positive tumors^[34]。 The synthesized Pc-LHRH has a drug entrapment efficiency of 20%w/w and exhibits NIR absorption(700 nm)and fluorescence emission(710 and 815 nm)necessary for efficient PDT treatment and FL imaging.And can be used for in vitro subcellular localization and in vivo organ distribution of Pc-LHRH based on the inherent fluorescence characteristics of the encapsulated Pc.As shown in Fig.4,the in vivo FL imaging showed that the tumor and liver had obvious FL imaging signals 10 hours after the tail vein injection of Pc-LHRH into the tumor-bearing mice,indicating that Pc was mainly enriched in the tumor and liver,showing its effect of targeting ovarian cancer xenograft tumor.In the absence of light and radiation,Pc-LHRH was almost non-toxic to human ovarian cancer A2780/AD cells in the studied concentration range of Pc(1.7~7.0μg/mL);However,after light irradiation,the A2780/AD cells treated with Pc-LHRH produced excessive reactive oxygen species,resulting in a significant（IC₅₀=0.9μg/mL）in PDT efficacy.The Pc-loaded functionalized dendrimer not only effectively improves many defects of the Pc fluorescent dye,but also realizes FL imaging at the cell and animal levels to track its distribution in subcells and tumor-bearing mice.The dual-functionalized tumor diagnosis and treatment reagent realizes drug delivery and photodynamic therapy mediated by NIR region FL imaging 。

Subsequently,Taratula et al.Encapsulated silicon naphthalocyanine(SiNc)into the fifth generation(G5)PPI dendrimer(PPI G5),and then modified its surface with biocompatible polymer PEG to construct the functionalized dendrimer nanosystem,which converted SiNc into biocompatible nanoplatform SiNc-NP^[27]。 The PPI-based dendrimer hydrophobic cavity effectively encapsulates and separates single SiNc molecules,reduces the aggregation of SiNc,improves the water solubility of SiNc-NP,and improves the efficiency of NIR FL imaging,PDT treatment and photothermal(PTT)treatment of ovarian cancer.When the laser power density was changed from 0.3 to 1.3 W·cm⁻²,the therapeutic mechanism of SiNc-NP could be switched from PDT to PDT-PTT combination therapy.And under NIR light irradiation(785 nm,1.3 W·cm⁻²）),the SiNc-NPs showed good heat generation ability(ΔT=40°C)and effectively generated reactive oxygen species necessary for PTT and PDT,respectively,without releasing SiNc from the nanoplatform.In addition,SiNc-NP showed significant NIR FL imaging performance in tumor-bearing mice,and there was no autofluorescence interference at the tumor site.The most important thing is that the fluorescence signal of the tumor site of tumor-bearing mice treated with SiNc-NP did not decrease after 785 nm laser（1.3 W·cm⁻²）irradiation for 10 min,indicating that SiNc is not prone to fluorescence quenching under the protection of PPI G5,and SiNc-NP has superior in vivo photostability,which can be used for long-term FL imaging guided tumor PDT and PTT treatment 。

Although fluorescent dye end-group modification at the periphery of dendrimers is the most common way to construct FL imaging agents,this often reduces dendrimer peripheral modification sites and affects the interaction of functionalized dendrimers with cells.Researchers have found that some fluorophores such as PDI and FITC can be embedded into the core of dendrimers to exert their fluorescence characteristics^[35,38]。 The method can effectively avoid the occupation of dendrimer terminal sites by fluorescent dye molecules.Cong et al.Employed tetraamine-modified PDI（PDI-NH₂）with fluorescent properties as a nucleating compound to react with N-tert-butoxycarbonyl(N-Boc)-protected and pentafluorophenol-activated lysine ester(Boc-Lys(Boc)-OPFP),followed by deprotection with trifluoroacetic acid,and repeated the process to prepare the fifth generation of PDI-cored polylysine dendrimer(PDI-PLL-G5)with fluorescent properties^[35]。 Finally,different amounts of N-hydroxysuccinimidyl ester-amidocaproic acid-biotin(NHS-LC-B)or NHS-polyethylene glycol-biotin(NHS-PEG-B)with targeting function were linked to PDI-PLL-G5 by amidation reaction,and the remaining amino groups on the surface of PDI-PLL-G5 were acetylated to obtain biotinylated PDI-PLL G5(G5-LC-5B,G5-PEG-1B,G5-PEG-3B,and G5-PEG 5B).in addition,the researchers acetylated the surface amino group of PDI-PLL-G5 without biotin modification to obtain acetylated PDI-PLL-G5(G5-Ac).Based on the fluorescence properties of the PDI core,the ability of biotin-conjugated PDI-PLL-G5 to be taken up by mouse breast cancer 4T1 cells can be monitored.the fluorescence signal of 4T1 cells incubated with G5-PEG-1B was stronger than that of 4T1 cells incubated with non-targeting G5-Ac,and the fluorescence brightness of 4T1 cell incubated with G5-PEG-5B was significantly higher than that of G5-PEG-1B and G5-PEG-3B,indicating that G5-PEG-3B was taken up most by 4T1 cell under the same condition.in comparison,the fluorescence signal of G5-LC-5B-treated 4 T1 cells was weak,indicating that the introduction of LC-B did not promote the uptake of PDI-PLL-G5 by 4T1 cells.Based on the fluorescence characteristics of PDI,the effect of biotinylated PDI-PLL-G5 on in vivo targeted tumor FL imaging in 4T1 tumor-bearing mice was subsequently evaluated.It was found that G5-PEG-5B,G5-PEG-3 B,G5-PEG-1B,G5-LC-5B and G5-Ac were injected into the tail vein of 4T1 tumor-bearing mice for 1 H,and all groups showed strong fluorescence signals in 4T1 tumor-bearing mice.the fluorescence signal in G5-Ac,G5-LC-5B,and G5-PEG-1B treated tumor-bearing mice gradually decreased with time.in contrast,the fluorescence signal in tumor-bearing mice treated with G5-PEG-3 B and G5-PEG-5 B stayed in the body for a longer time,and the fluorescence signal in the tumor gradually increased over time(Figure 5A).in vivo FL imaging experiments demonstrated that increasing the amount of PEG-B modification in PDI-PLL-G5 increased the accumulation of PDI-PLL-G5 at the tumor site,thereby mediating a high intensity fluorescence signal.the accumulation of biotinylated PDI-PLL-G5 in 4T1 tumors was further quantified by quantitative analysis of tumor regional fluorescence intensity.the fluorescence intensity of tumors in G5-Ac,G5-LC-5B and G5-PEG-1B groups decreased with time,but the fluorescence signal of tumors in G5-PEG-3 B and G5-PEG-5 B groups increased with time at the beginning of injection.the fluorescence signal of G5-PEG-3B group decreased rapidly with time after 8 H.However,the fluorescence intensity of tumors in G5-PEG-5 B group continued to increase within 24 H,and the fluorescence intensity at 24 H was 2 to 3 times higher than that in other groups(Fig.5B).the tumor-to-normal tissue fluorescence signal ratio(TNR)of tumor-bearing mice in the G5-PEG-5B group was significantly increased after injection and reached about 3.5 at 24 H.in contrast,the TNR of other groups was in the range of 0.9∼2.0(Figure 5C).This further confirmed that modification of PDI-PLL-G5 with PEG-B improved the in vivo tumor targeting ability of PDI-PLL-G5.the functional dendrimer constructed by the fluorescent PDI core can evaluate the in vivo biodistribution and tumor enrichment of PEG-B PDI-PLL-G5 through in vivo and in vitro FL imaging.the most important thing is that G5-PEG-5B has the specificity of targeting 4T1 cells and 4T1 tumors,and can provide lasting and stable FL imaging signals for immediate and sensitive fluorescence monitoring。

Bukun et al.Synthesized a series of PAMAM dendrimers(FITC-G0~FITC-G5)of different generations with fluorescent dye FITC as the core by repeated Michael addition reaction with ethylenediamine and further amidation reaction with methyl acrylate^[38]。 Then an excess of antimetabolite antitumor drug 5-fluorouracil(5-FU)was mixed with the above synthesized PAMAM dendrimer with FITC as the core,and 5-FU was loaded into the dendrimer FL imaging reagent.Based on the fluorescence properties of this dendrimer conferred by the FITC core,the uptake of 5-FU-loaded PAMAM dendrimer(G0-G5)by human gastric cancer AGS cells can be monitored by fluorescence microscopy.After AGS cells were treated with 5-FU-loaded FITC-cored dendrimer for 4 H,cell internalization was observed by FITC-mediated FL imaging under a fluorescence microscope.the results showed that the internalization of the functionalized dendrimer increased from 20.00%±2.45%of FITC-G0 to 39.00%±0.82%of FITC-G5 with the increase of dendrimer generations,indicating that the cellular uptake and internalization increased with the increase of dendrimer generations.the internalization of this functionalized dendrimer by AGS cells treated with 5-FU-loaded FITC-G2 and FITC-G3 for 4 H was 22.27%±1.68%and(25.00%±1.22%),respectively.the proportion of FITC-G4(31.47%±0.41%)internalized into cells was significantly higher than that of FITC-G3(25.00%±1.22%),and the proportion of FITC G5(39.00%±0.82%)internalized into cells was significantly higher than that of FITC G4(31.47%±0.41%).FL imaging results showed that PAMAM dendrimers were taken up by AGS cells in a algebra-dependent manner,thereby facilitating the delivery of 5-FU into the cells.the PAMAM dendrimer with FITC as the core can carry out efficient delivery of 5-FU while carrying out FL imaging of cancer cells,and the delivery efficiency of the functionalized dendrimer to 5-FU is analyzed by using the intensity of a fluorescence signal。

the dendrimer loaded with fluorescent dye molecules constructed so far can realize FL imaging at cell and animal levels.and the fluorescence signal and the image resolution of the dendrimer are better than those of the dendrimer autofluorescence.More importantly,the good water solubility and physicochemical properties of dendrimers can significantly improve the water solubility and photostability of fluorescent dye molecules,which can achieve long-term FL imaging and have good biological safety in vivo.However,in the dendrimer loaded with fluorescent dye molecules,the fluorescent dye molecules are loaded into the dendrimer in the form of physical wrapping,due to the insufficient interaction force between the functionalized dendrimer and the fluorescent dye,fluorescent dye molecules are easy to leak before reaching the tumor site,and the loading rate of fluorescent dye molecules in dendrimers is low,resulting in low FL imaging signal mediated by low-dose FL imaging reagents.Therefore,how to enhance the interaction force between dendrimers and fluorescent dyes and how to improve the loading rate of fluorescent dye molecules in dendrimers still need further study。

Common inorganic fluorescent nanoparticles mainly include fluorescent quantum dots(QDs)and upconversion nanoparticles(UCNPs)^[42,43]。 As a new generation of fluorescent markers,QDs can show different wavelengths of fluorescence under ultraviolet irradiation.Compared with organic fluorescent dyes,QDs are easier to resist photobleaching for a long time due to their smaller particle size,narrower fluorescence emission peak,higher quantum yield and brightness^[44]。 At present,the common QDs are mainly silver chalcogenide（Ag₂X;X=S,Se,Te)QDs,cadmium selenide/zinc sulfide(CdSe/ZnS)QDs,and carbon dots(CDs )^[42,45]。 QDs have longer emission lifetime and stronger fluorescence signal intensity.For example,the fluorescence emission intensity of single CdSe QDs is 20 times that of rhodamine,but the bleaching rate is only one percent of rhodamine^[46]。 Based on high photostability,detection sensitivity and light penetration depth,UCNPs are used as excellent FL imaging reagents in biomedicine.Its main component is rare earth element doped inorganic matrix,such as lanthanide oxide（Ln₂O₃）etc^[43,47]。 the energy level structure of rare earth elements determines their ability to convert two or more long-wavelength low-energy near-infrared photons into short-wavelength high-energy photons through nonlinear radiation,which can significantly enhance The light penetration depth into tissues,so FL imaging has high sensitivity^[43]。 Although QDs and UCNPs have excellent optical properties,their potential cytotoxicity,poor water solubility,and lack of tissue specificity limit their in vivo FL imaging applications^[42,43]。 the combination of dendrimers and inorganic fluorescent nanoparticles can make up for many defects of inorganic fluorescent nanoparticles,and can achieve efficient FL imaging in vivo and in vitro,which has been widely used in The field of cancer diagnosis and treatment in recent years。

Silver chalcogenide（Ag₂X;X=S,Se,Te)QDs,as narrow-gap semiconductor nanocrystals,have high stability and high quantum yield,and have unique NIR-II FL imaging properties,which are widely used in biomedical diagnosis of cancer^[48]。 Awasthi et al.Prepared silver sulfide quantum dot（Ag₂S QDs）with fluorescence properties,and combined it with PEGylated polyacylthiourea dendrimer(PEG-PATU)to obtain PEG-PATU encapsulated Ag₂S QDs（PEG-PATU Ag₂S QDs）^[49]。 The silver nitrate（AgNO₃）is added to the PEG-PATU solution by a one-pot method,and the PEG-PATU promotes the absorption of silver ions into the dendrimer core through coordination with thiourea groups.Subsequently,the addition of sodium sulfide（Na₂S）triggers the in situ formation of Ag₂S nanocrystals in PEG-PATU nanocages,culminating in the formation of PEG-PATU Ag₂S QDs.Based on the fluorescence characteristics of PEG-PATU Ag₂S QDs,cell imaging observation can be carried out by fluorescence microscope.After the lung cancer A549 cells were incubated with PEG-PATU Ag₂S QDs for 4 H,the fluorescence intensity of the cells increased with the increase of PEG-PATU Ag₂S QDs concentration and tended to be stable with the extension of incubation time,which indicated that A549 cells had successfully taken up PEG-PATU Ag₂S QDs.In vivo cell fluorescence tracing with A549 cancer cells labeled with PEG-PATU Ag₂S QDs could further explore the primary localization and secondary distribution of metastatic tumor cells in the blood.A549 cells labeled with PEG-PATU Ag₂S QDs were injected into the tail vein of BALB/C mice under 808 nm laser（45 mW·cm⁻²）irradiation,and then NIR-Ⅱregion FL imaging was performed at different time points.The results showed that the fluorescence signal appeared in the liver at 2 min after injection,but the signal gradually disappeared within 24 H,while the other tissues in vivo gradually became bright,indicating that A549 cells were redistributed throughout the body.As a control,the same dose of PEG-PATU Ag₂S QDs was intravenously injected into healthy BALB/C mice,and FL imaging was performed under the same conditions.The results showed that the FL imaging signal mediated by PEG-PATU Ag₂S QDs was mainly confined to the liver,while only a weak fluorescence signal was observed in other parts of the mouse,indicating that most of the PEG-PATU Ag₂S QDs was localized in the liver.The above results indicate that PEG-PATU Ag₂S QDs has excellent NIR-II FL imaging performance in vivo,which can be used to efficiently label and track the migration rate of A549 cancer cells in vivo,and depict the behavior of A549 cells in vivo to provide early in vivo diagnostic information for lung cancer metastasis 。

Based on the narrow emission bandwidth,high fluorescence quantum yield,and long fluorescence lifetime,CdSe/ZnS QDs are often used as fluorescent probes for monitoring cancer cells and for early FL imaging of tumors^[50]。 Li et al.Used CdSe/ZnS QDs with fluorescence characteristics as a template,and successively coordinated lipoic acid(LA)and hydrazine（NH₂NH₂）functionalized dendrimers（LA-Glu(G2)-NHNH₂）,LA-methoxylated polyethylene glycol(LA-mPEG),RGD-polyethylene glycol-LA(RGD-PEG-LA)on its surface to obtain CdSe/ZnS QDs loaded RGD-targeted PEGylated dendrimers(T-PHDs )^[51]。 Finally,DOX was coupled to the surface of LA-Glu(G2)-NHNH₂in T-PHDs via an acid-sensitive hydrazone bond to give RGD-targeted DOX-coupled PEGylated dendrimers(T-DPHDs)loaded with CdSe/ZnS QDs(Figure 6A).Meanwhile,DOX-coupled PEGylated dendrimers(DPHDs)loaded with CdSe/ZnS QDs without modification by targeting reagent RGD-PEG-LA were also synthesized.Based on the intrinsic fluorescence of CdSe/ZnS QDs,T-DPHDs-mediated FL imaging can be used to monitor the dynamic changes of primary tumors and metastases in real time.According to the in vivo FL imaging of 4T1 tumor-bearing mice,compared with DPHDs at the same time point after injection,T-DPHDs modified with targeting molecule RGD showed faster fluorescence signal and stronger brightness at the tumor site.In vitro tumor FL imaging at 24 H after injection showed that the tumors of tumor-bearing mice treated with DPHDs and T-DPHDs had fluorescence signals,and the fluorescence signals of tumor-bearing mice treated with T-DPHDs were higher(Figure 6B).This suggests that the active targeting ability of RGD allows T-DPHDs to accumulate at a faster rate and in greater amounts at the 4T1 tumor site.Therefore,T-DPHDs can be used as targeted fluorescent probes to monitor primary tumors withαvβ3 integrin overexpression.Breast cancer metastasis was then monitored by FL imaging of the lungs of the tumor-bearing mice.The results showed that lung tumor metastasis was clearly seen in the bright field images in vitro after intravenous injection of saline,DPHDs and T-DPHDs into 4T1 tumor-bearing mice.By evaluating the fluorescence distribution in the isolated lung tissue and 4T1 primary tumor of tumor-bearing mice treated with DPHDs and T-DPHDs,it can be seen that DPHDs showed non-specific fluorescence signals in the lung,while T-DPHDs only showed high-intensity fluorescence signals in the lung and primary 4T1 tumor,which means that T-DPHD has the potential to monitor tumor metastasis and can monitor the dynamic situation of tumor metastasis through fluorescence signals(Fig.6C).The functional dendrimer-loaded CdSe/ZnS QDs improves the water solubility of the CdSe/ZnS QDs through the introduction of the dendrimer,can endow T-DPHDs with targeting specificity through the functional modification of the dendrimer,and can realize the effective monitoring of primary tumors and metastatic tumors withαvβ3 integrin overexpression by utilizing the fluorescence characteristic of the CdSe/Zn QDs 。

CDs are a subset of quantum dots with a size of less than 10 nm,which have strong luminescence and small size characteristics similar to traditional quantum dots,and have many functional groups on their surfaces,which make them easy to modify and couple,so that CDs can be combined with a variety of nanomaterials to exert their fluorescence characteristics^[52]。 Ma et al.Covalently bonded CDs with peptide dendrimers and loaded DOX to construct a tumor diagnosis and treatment system^[53]。 Blue fluorescent CDs were prepared by microwave pyrolysis using glucose as carbon source and mixed with polyethylene glycol 200(PEG200),and then modified with amino-disulfide-amino（NH₂-S-S-NH₂）by amidation reaction to obtain thiolated CDs(CDs-SH).Subsequently,the first generation peptide dendrimer(D-S-S-D)prepared by the condensation of N-tert-butoxycarbonyl-N'-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine and L-lysine methyl ester hydrochloride was covalently bonded to CDs-SH via thiol oxidation reaction,and DOX was adsorbed on the surface of CDs to obtain the DOX-loaded peptide dendrimer-modified CDs complex(CD-D/DOX).Finally,CD-D/DOX was encapsulated by zwitterionic polycarboxybetaine methacrylate(pCBMA)to obtain zwitterionic functionalized and DOX-loaded peptide dendrimer-encapsulated CDs complex(pCBMA(CD-D/DOX)).The hydrophilicity and low interfacial energy of pCBMA can reduce the nonspecific protein adsorption of pCBMA(CD-D/DOX).At the same time,in the tumor microenvironment(pH<6.8),the surface charge of the constructed pCBMA(CD-D/DOX)can be reversed from a negative value(−1.62 mV)at pH 7.4 to a positive value(+7.65 mV),which can interact with the negatively charged cancer cell membrane and improve its uptake by cancer cells.The excellent fluorescence characteristics of CDs endow pCBMA(CD-D/DOX)with biofluorescence imaging function,and the uptake ability of pCBMA(CD-D/DOX)in vitro cancer cells can be evaluated by confocal microscopy.After 4T1 cells were incubated with pCBMA(CD-D/DOX)for 2 H,obvious CDs blue fluorescence signals were observed in the cytoplasm,and obvious CDs fluorescence signals were observed in the cytoplasm and nucleus when the incubation time was extended to 6 and 16 H,and the CDs(blue)and DOX(red)fluorescence in 4T1 cells overlapped at different incubation time points,and the CDs-mediated fluorescence signals could trace the distribution of pCBMA(CD-D/DOX)in 4T1 cells(Fig.7).This indicates that DOX-loaded pCBMA(CD-D/DOX)can be internalized by 4T1 cells and enter the nucleus to exert anticancer effects in vitro.The tissue distribution and metabolism of pCBMA(CD-D/DOX)could be assessed by CDs-mediated FL imaging in vivo,and no fluorescence signal from CDs was found in the tumor in vitro after treatment with pCBMA(CD-D/DOX)or free CDs for 8 H,indicating that pCBMA[CD-D/DOX]could be safely metabolized out of the body.The constructed pCBMA(CD-D/DOX)can not only trace anticancer drugs in vitro through CDs-mediated FL imaging,but also monitor the distribution and metabolism of the vector in tumor-bearing mice 。

Similarly,Li et al.Synthesized yellow fluorescent CDs(y-CDs)by one-step hydrothermal treatment using 4-aminosalicylic acid(4-ASA)as precursor^[54]。 At the same time,P-glycoprotein(P-gp)inhibitor polyethylene glycol 1000 vitamin E succinate(TPGS)was modified on the surface of G5 PAMAM dendrimer to form G5-TPGS conjugate.Then,the G5-TPGS and Y-CDs were partially covalently coupled and partially electrostatically adsorbed to form a G5-TPGS-encapsulated Y-CDs complex(G5-TPGS@Y-CDs),and DOX was physically encapsulated into the G5-TBPS@Y-CDs to obtain(G5-TBPS@Y-CDs)-DOX.the yellow fluorescence emitted by y-CDs can be used for in vitro cancer cell tracking of G5-TPGS@y-CDs.in vitro cancer cell FL imaging showed that after incubation of human breast cancer doxorubicin-resistant MCF-7/ADR cells for 6 H,the fluorescence of y-CDs(yellow)and DOX(red)could be observed simultaneously in(G5-TPGS@y-CDs)-DOX-incubated MCF-7/ADR cells compared with PBS-incubated MCF7/ADR cells,and the fluorescence signal mediated by y-CDs could indicate the presence of(G5-TPGS@y-CDs)-DOX in MCF7/ADR cell.This indicates that the G5-TPGS@y-CDs complex can be loaded with DOX and delivered to MCF-7/ADR cells to achieve efficient enrichment of anticancer drugs in drug-resistant cancer cells.in addition,the fluorescence signal presented by y-CDs can further evaluate the effect of ultrasound-targeted microbubble destruction(UTMD)technology on the enrichment of(G5-TPGS@y-CDs)-DOX in tumor sites in vivo.in vivo FL imaging tracking showed that after 6 H of intravenous injection,fluorescence signals were observed in the tumor sites of(G5-TPGS@y-CDs)-DOX-treated and(G5-TPGS@y-CDs)-DOX+UTMD-treated tumor-bearing mice compared with those of y-CDs-treated tumor-bearing mice,and the strongest fluorescence signals were observed in the tumor sites of(G5-.the fluorescence signal mediated by y-CDs can be used to monitor the distribution of(G5-TPGS@y-CDs)-DOX in vivo during tumor therapy,and as a new fluorescent probe,it is expected to achieve FL imaging-guided chemotherapy of drug-resistant tumors。

Lanthanide-doped nanoparticles,as the most common UCNPs,have the advantages of deep tissue penetration,no background fluorescence noise interference,and high imaging sensitivity,and are used as FL imaging reagents with excellent performance in the field of cancer diagnosis and treatment^[55,56]。 Mekuria et al.Synthesized dysprosium(Dy),erbium(Er),and terbium(Tb)rare earth element doped lanthanide oxide Ln₂O₃nanoparticles(NPs)with fluorescent properties by one-pot method^[47]。 After that,the 4.5-generation PAMAM dendrimer(G4.5)was wrapped around Ln₂O₃NPs（Ln=Dy,Er,and Tb)by electrostatic adsorption to obtain G4.5-wrapped Ln₂O₃NPs（G4.5@Dy,Er,Tb oxide NPs）.Meanwhile,folic acid(FA),a small molecule compound,was coated on the surface of Ln₂O₃NPs in the same way to prepare a comparative material FA-coated Ln₂O₃NPs（FA@Dy,Er,Tb oxide NPs）（(Fig.8 a).By analyzing the fluorescence signals of G4.5@Dy,Er,Tb oxide NPs and FA@Dy,Er,Tb oxide NPs after incubating cervical cancer HeLa cells,the in vitro HeLa cell uptake ability of G4.5 dendrimer and FA-coated Tb oxide NPs could be evaluated.FL imaging of HeLa cells in vitro showed that after 24 H of incubation,both HeLa cells incubated with G4.5@Dy,Er,Tb oxide NPs and HeLa cells incubated with FA@Dy,Er,Tb oxide NPs had Tb oxide NPs green)and Tb oxide NPs red)fluorescence signals,and G4.5@DyThe fluorescence signals of Tb oxide NPs and Tb oxide NPs in HeLa cells incubated with Tb oxide NPs were significantly stronger than those in HeLa cells incubated with FA@Dy,Er,Tb oxide NPs,which was attributed to the higher water solubility of PAMAM dendrimer compared with FA,so that G4.5@Dy,Er,Tb oxide NP was taken up more by HeLa cells and showed enhanced fluorescence signals(Fig.8B).In addition,the paramagnetism of Dy⁺³and Tb⁺³can be used for T2-weighted MR imaging of cancer cells in vitro,G4.5@Dy,Er,The Tb oxide NPs relaxation rate of Tb oxide NPs,Tb oxide NPs,is significantly larger than the r₂relaxation rate of FA@Dy,Er,Tb oxide NPs,（8.37 mM⁻¹·s⁻¹）,with higher T2-weighted MR imaging performance.The constructed G4.5@Dy,Er,Tb oxide NPs can be used for FL/MR dual-modality imaging of cancer cells based on lanthanide-mediated upconversion fluorescence properties and paramagnetism.Most importantly,the introduction of dendrimers endows G4.5@Dy,Er,Tb oxide NPs with strong water solubility,which can realize HeLa cell lifting FL imaging and can be used for dual-modality imaging monitoring of early cancer cells 。

Due to the introduction of the functional dendrimer,the inorganic nano fluorescent particle loaded with the dendrimer constructed at present improves the water solubility of the inorganic nano fluorescent particle,improves the biocompatibility thereof,and is modifiable based on the dendrimer,the surface of the FL imaging reagent can be modified with functional reagents such as polyethylene glycol,a targeting reagent,zwitterions and the like to improve the blood circulation time of the FL imaging reagent in vivo and the aggregation at a tumor,and shows sensitive and excellent FL imaging performance in vivo and in vitro.However,the application of dendrimer-loaded inorganic fluorescent nanoparticles in vivo is still in the early experimental stage.Low concentrations of dendrimer-loaded inorganic fluorescent nanoparticles do not have significant pathological reactions when used for FL imaging in vivo,but their long-term toxicity in vivo is still unclear.Therefore,the biological toxicity assessment of dendrimer-loaded inorganic fluorescent nanoparticles for long-term application in vivo still needs to be further studied to provide a reference for exploring dendrimer-loaded inorganic fluorescent nanoparticles with longer fluorescence lifetime and higher in vivo biosafety。