Organelle imaging with carbon dots: strategies, challenges, and perspectives

Abstract

Organelle imaging is an efficient approach to gain information about intracellular events and dynamics of subcellular structures. In this case, carbon dots (CDs) are outstanding fluorescent probes for organelle imaging due to their excellent biocompatibility, tunable and stable fluorescence, anti-photobleaching, and easy and cheap preparation. In the past decade, numerous investigations have made great progress in regulating the physicochemical properties of CDs for targeted organelle imaging. However, there are several obstacles that hinder the further understanding of subcellular events, such as the unsatisfactory organelle specificity of CDs and their inconsistent organelle-targeting mechanism. In addition, researchers have focused on how to produce organelle-targeting CDs but ignored the fact that cells and organelles also affect the subcellular distribution of CDs. Thus, in this review, we outline the development in the field of organelle-targeting imaging using CDs as probes, summarize the general strategies for targeted imaging using CDs, discuss the challenges in this field, and propose potential solutions. We hope that this review will facilitate the further development of organelle imaging using CDs as probes.

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1. Introduction

Most eukaryotic cells contain a nucleus, lysosomes, Golgi apparatus, and other organelles, where biochemical reactions occur to keep cells alive. Accordingly, if one of these organelles is damaged and the related reactions are abnormal, the cell becomes apoptotic and diseases occur.¹ In this case, the visualization of the individual compartments and reactions in different organelles or subcellular structures facilitates the further understanding of the cell status, metabolism, functionality, diseases and treatments,^2,3 where fluorescence probes are vital tools to achieve visualization or imaging. To date, several types of fluorescent probes, such as organic dyes, fluorescent proteins, and fluorescent nanoparticles, have been developed and employed for the visualization of cells.^4–7 However, the photobleaching of organic dyes, the complicated synthetic procedure for fluorescence proteins, the toxicity of semiconductor quantum dots, the high cost of upconversion nanoparticles, and other drawbacks make it necessary to develop new organelle imaging probes to avoid these disadvantages.

Carbon dots (CDs) show great potential application in the fluorescence imaging of organelles due their favourable fluorescence, high photo- and chemical-stability, excellent biocompatibility, low toxicity, easy preparation, tunable organelle targeting, and high resolution.^8–11 Wang¹² and Yang¹³ were the first researchers to observe that CDs are distributed mainly in the cytoplasm. Li investigated the distribution of CDs in HeLa, SMCC-7721, and HEK293 cells using fluorescence images and TEM images and found similar results.¹⁴ Karthik first reported that quinolone-tethered CDs were distributed in both the cytoplasm and nucleus.¹⁵ Datta prepared cationic quaternized CDs that could label the cell nucleus and reported that the distribution of CDs could be regulated by combining them with anionic graphene oxide.¹⁶ Since then, increasing research has focused on imaging the nucleus and nucleolus using different carbon dots.^17–28 Meanwhile, the fluorescence imaging of the mitochondrion,^29–38 lysosome,^39–46 endoplasm reticulum,^47–50 Golgi apparatus^51–55 and lipid drops^56–61 using CDs has also witnessed great developments. Recent reviews summarized the progress in individual organelle imaging, highlighting the properties, precursors, and sensing applications of CDs.^62–64 However, to date, there are still no straightforward strategies for organelle targeting using CDs, the interactions between CDs and organelles are not clear, and the organelle-targeting mechanism of CDs is controversial. Furthermore, a review addressing these issues is still lacking.

In this review, we focus on the key issues associated with the use of CDs as organelle imaging probes. Initially, we summarize the general strategies for organelle imaging using CDs, including the intrinsic targeting ability of one-pot CDs, post-modification with specific species, and uptake pathways of CDs. Then, we outline the challenges in preparing “ideal” CDs probes and investigating their targeting mechanism, including their unsatisfactory organelle specificity and lack of CDs targeting less-concerned organelles. Subsequently, we suggest potential solutions, where we highlight the purification and intracellular transport of CDs, the interaction between CDs and intracellular molecules, and the super-resolution imaging techniques.

2. The general strategies for organelle targeting

Various studies have demonstrated that the size,¹⁵ hydrophilicity/hydrophobicity,¹¹ functional groups,⁴² surface charge,⁶⁵ and chirality⁵¹ of CDs are vital for their subcellular distribution. However, there is still a lack of information on how to achieve specific organelle targeting. Besides, clear and consistent elucidation of the organelle-targeting mechanism of CDs is still challenging. Herein, we summarize the generally used strategies for organelle targeting using CDs, which mainly include intrinsic organelle targeting, post-modification with specific species, and use of the uptake pathway. We believe that this summary will provide readers with an updated understanding of the regular patterns in the targeting feature of CDs.

2.1 Intrinsic organelle-targeting CDs

Some carbon dots (CDs) without special post-modification can target certain organelles naturally, which are defined as intrinsic organelle-targeting CDs. Generally, their targeting ability is attributed to the surface groups, zeta potential, hydrophilicity/hydrophobicity, and other physical/chemical properties of CDs. One-pot-prepared CDs usually inherit some specific properties of their precursors,^47,63,66 and thus rationally selected precursors that can interact with specific organelles are vital to ensure the intrinsic organelle-targeting ability of CDs. The current widely used precursors can be classified into two categories, i.e., unspecific precursors without targeting nature and specific precursors with targeting features for certain organelles. Both types of precursors can produce CDs with intrinsic targeting ability.

2.1.1 CDs generated from unspecific precursors.
2.1.1.1 Lysosome targeting CDs. Typical examples of CDs prepared using unspecific precursors are lysosome-targeting CDs. The lysosome is the digestion organelle of the cell and contains abundant protons to maintain a low pH value, maintaining the activity of digestion enzymes. Imaging of the lysosome is beneficial to understand living processes such as autophagy, the digestion of external nutrient substances and generation of endogenous waste. Amino groups easily bind with protons, which is regarded as the weak alkaline or acidotropic effect of amino groups. Thus, the enriched amino groups on the surface of CDs are believed to be responsible for their lysosome-targeting ability. Ethanediamine,⁴⁴ tetraaminobenzene,⁴⁶ triethylenetetramine,⁶⁷ dopamine, 1,2-diaminobenzene,⁶⁸ spermine⁶⁹ and other amino-containing precursors have been successfully used to prepare amino-containing CDs to achieve lysosome-targeting ability. Huang's group proposed a functional preservation strategy (Fig. 1A) to prepare CDs at ambient temperature to avoid their decomposition and preserve the functional groups of the starting materials.⁴⁴ By simply mixing p-benzoquinone and ethylenediamine, the spontaneous exothermal reaction provided appropriate heat to form CDs and create rich amine groups on the CDs, facilitating lysosome-targeted imaging. This strategy is energy-saving. However, spontaneous exothermal reactions occur with limited precursors and the majority of precursors require high temperature to form CDs. Liu et al. prepared CDs by reacting dexamethasone and 1,2,4,5-tetraaminobenzene at 160 °C via a microwave-assisted hydrothermal method.⁴⁶ The as-prepared CDs were enriched with amine groups and could target lysosomes due to the acidotropic effect. Tong reported the synthesis of NH₂ group-functionalized CDs for long-term lysosome imaging by treating rose bengal and branched polyethyleneimine at 160 °C.⁷⁰ Similar methods can also be applied to prepare lysosome-targeting CDs by heating amino-containing starting materials at high temperature (130 °C–160 °C), and their targeting ability is attributed to the weak alkaline or acidotropic effect of the amino groups on their surface.^67,69,71,72 The typical process for the preparation of intrinsic lysosome-targeting CDs and lysosome imaging is presented in Fig. 1B and C, respectively.

Fig. 1 (A) Schematic representation of functional preservation strategy to prepare lysosome-targeting CDs and imaging of lysosomal pH, reprinted with permission from ref. 44, Copyright 2018, The Royal Society of Chemistry. (B) Confocal fluorescence images of MCF-7 cells treated with CDs prepared from spermine and rose bengal, reprinted with permission from ref. 69, Copyright 2021, Elsevier Ltd. (C) Schematic illustration of the preparation of lysosome-targeting CDs and their application in the detection of FA, reprinted with permission from ref. 46, Copyright 2019, The Royal Society of Chemistry.

Recently, Singh et al. obtained lysosome-targeting CDs by hydrothermal treating extracts of neem plant roots without clear identification of their composition. The authors speculated that the appropriate lipophilicity induced by the –COOH, C–O–C, and amino functionalities on the surface helped to achieve specific lysosomal localization.⁷³ A similar result was reported in another study, in which CDs with moderate lipophilicity were prepared via the hydrothermal carbonization of chloranil and triethylenetetramine, which targeted lysosomes within 30 min.⁷⁴ Zhi purified the raw products from the hydrothermal treatment of citric acid and urea in formamide with a C₁₈ reversed-phase column and obtained blue-emitting CDs (CD-B) and red-emitting CDs (CD-R), which targeted lysosomes/mitochondria and only lysosomes, respectively. The authors proposed that the difference in the hydrodynamic size of the CDs was responsible for the difference in their localization.⁷⁵ These studies show some inconsistencies with respect to the structure–activity relationship of lysosome-targeting CDs, but may also open up more potential ways to prepare lysosome-targeting CDs.

2.1.1.2 Nucleus- or nucleolus-targeting CDs. The nucleus or nucleolus contains genetic DNA, RNA and functional proteins, which are the control center of the cell. Nucleus- or nucleolus-targeting CDs can also be generated from unspecific precursors. The most characteristic substances in the nucleus and nucleolus are DNA and RNA, which are complex macromolecules with negative charges. Accordingly, CDs with highly positive charges originating from various organic amine molecules have been successfully prepared and used for nucleus/nucleolus imaging.

Datta obtained cationic quaternized carbon dots by heating a mixture of tris and betaine, which possessed a zeta potential as high as +53 mV.¹⁶ The strong positive charge facilitated the interaction between the CDs and DNA/RNA, resulting in the localization of the CDs in the nucleus. Several later studies also reported the preparation of nucleus/nucleolus-targeting CDs with positive-charged surfaces using dopamine/ethylenediamine,¹⁹p-phenylenediamine/Ni²⁺,²⁷ 1,5-diaminonaphthalene,⁷⁶ citric acid/PEI,⁷⁷m-phenylenediamine/p-aminobenzoic acid,⁷⁸ and m-phenylenediamine/aminourea hydrochloride.⁷⁹ The interaction between CDs and DNA/RNA was identified by enzymolysis experiments, in which CDs could stain only the nucleus or nucleolus after the cells were treated with RNase or DNase.^27,80–83

Interestingly, negatively charged CDs can also interact with DNA/RNA and target the nucleus/nucleolus.^{22,23,28,84,85} Hua et al. attributed the RNA affinity to the specific surface chemistry of the CDs (Fig. 2A).²³ Li suggested that there is non-covalent interaction between the negatively charged CDs and nucleic acid because of the increase in fluorescence upon the addition of DNA or RNA.²⁸ Liu proposed that the hydrogen bonding of the CDs with DNA/RNA facilitated the nuclear targeting (Fig. 2B).⁸⁶ Given that hydroxyl/carboxyl groups commonly remain on the surface of CDs, we believe that the hydrogen bonding explanation is more reasonable. Kong suggested that the protonation of the oxygen groups in CDs facilitates their interaction with the alkaline chromatin, which may result in their nucleus-targeting ability.⁸⁷ Besides positively/negatively charged CDs, zwitterionic CDs can also target the nucleus or nucleolus due to their zwitterionic surface.^18,25,88 It is noteworthy that most of these investigations addressed the importance of small-size CDs for nuclear targeting because large CDs would be excluded by the nuclear pore complex.

Fig. 2 (A) Schematic illustration of the preparation of CDs for nucleus imaging and the interaction between CDs and DNA, reprinted with permission from ref. 23, Copyright 2018, the American Chemical Society. (B) Presentation of the preparation of negative-charged CDs with intrinsic nuclear imaging ability due to the hydrogen bond, reprinted with permission from ref. 86, Copyright 2019, The Royal Society of Chemistry. (C) Schematic illustration of the preparation of nucleus-targeting CDs and their application in multiple cell lines, reprinted with permission from Ref. 82, Copyright 2022, Elsevier Ltd. (D) Illustration of the preparation of nucleus-targeting CDs from biomass, reprinted with permission from ref. 90, Copyright 2020, the American Chemical Society.

Different from the above-mentioned studies in which CDs were obtained at high temperatures, Choi et al. prepared a type of nucleus-targeting CDs at room temperature via an acid-assisted carbonization method by treating the product of C/C₁₂-PEG-g-PDMA and 1,3-propane sultone.⁸⁹ The surface of the obtained CDs contained zwitterionic dodecane/sulfobetaine groups. The response to a variation in pH by these zwitterionic groups facilitated the transportation of the CDs from the cytosol to the nucleus in cancer cells, but not in normal cells. Therefore, it is promising to develop a cancer diagnostic tool that can differentiate normal cells from cancer cells using these CDs.

Besides organic molecules, natural materials without certain structures such as hydrogenated rosin could also be used to produce nucleus-localized CDs (Fig. 2D).⁹⁰ The CDs could be used to detect Fe³⁺ with a detection limit of 6.16 μM.

2.1.1.3 Mitochondria-targeting CDs. Mitochondria are dual-layer-phospholipid-membrane organelles with a highly negative membrane potential (mitochondria membrane potential, MMP), where ATP is generated to provide energy for multiple processes, such as cell division and signal transduction. A widely validated targeting mechanism for mitochondria is the electronic interaction between mitochondria and probes.

Wu's group prepared mitochondrial-targeting CDs via the one-step hydrothermal treatment of mercaptosuccinic acid, ethylenediamine, and chitosan.³² The intrinsic mitochondria-targeting ability was attributed to the sufficient delocalization, lipophilicity, and positive charges of the CDs. The CDs were used to achieve mitochondria-targeted photodynamic therapy (Fig. 3A).³² They also reported the preparation of CDs using glycerol and silane to target mitochondria due to the presence of cationic species on the CDs. The CDs were responsive to polarity, and thus used to differentiate cancerous cells from normal cells.³³ In another study, mitochondria-targeting single-layered graphene quantum dots (sGQDs) were prepared via the hydrothermal treatment of perylene tetracarboxylic anhydride and polyethylenimine.³⁶ The sGQDs localized in the mitochondria selectively and the positive charges and lipophilic features of sGQDs were responsible for the organelle targeting. Several subsequent studies also attributed the intrinsic targeting ability of CDs to their positive surface charges and the electronic interaction between CDs and mitochondria (Fig. 3B).^91–94 However, it is noteworthy that this electronic interaction mechanism is not specific. CDs with positive charges on their surface can be localized in mitochondria and endoplasm reticulum, or mitochondria and nucleolus, simultaneously.^71,95,96 Besides, negatively charged CDs decorated with citric acid could also target mitochondria, which was driven by the affinity between citric acid and mitochondria.⁹⁷ If this affinity can be validated widely, there will be another approach towards intrinsic mitochondria-targeting CDs.

Fig. 3 (A) Schematic illustrating the synthetic route of CDs and their applications as a fluorescent mitochondrial-tracker, reprinted with permission from ref. 32, Copyright 2017, The Royal Society of Chemistry. (B) Preparation of self-targeting CQDs and their application for mitochondrial ONOO⁻ imaging, reprinted with permission from ref. 93, Copyright 2021, the American Chemical Society. (C) Dynamic lipid droplet coalescence observed from fluorescent imaging results with CDs as probes, reprinted with permission from ref. 99, Copyright 2023, Elsevier Ltd. (D) CLSM images of co-localization with different organelles from an ER-targeting CDs, reprinted with permission from ref. 100, Copyright 2020, The Royal Society of Chemistry.

2.1.1.4 Lipid droplet- and endoplasmic reticulum-targeting CDs. Lipid droplets (LD) are the storage organelles of neutralized lipids, while the endoplasmic reticulum (ER) is the location for the synthesis of proteins and all types of lipids. Thus, targeting lipid droplets and the endoplasmic reticulum can facilitate the understanding of the synthesis and metabolism of lipids and proteins. Both LD and ER are highly lipophilic. Therefore, CDs prepared from hydrophobic materials and are lipophilic to some extent are expected to be LD-targeting and ER-targeting.

Liu prepared two novel types of amphiphilic LD-targeting CDs via the one-step treatment of o-phenylenediamine at room temperature and 200 °C.⁵⁶ The CDs achieved long-term tracking of LDs, and thus were successfully applied for the real-time monitoring of lipid catabolism and pharmacodynamic evaluation of lipid-lowering drugs. Wang prepared LD-targeting CDs via the hydrothermal treatment of 4-piperidinoaniline at 160 °C in ethanol.⁶¹ After a comparative experiment with commercial LD-targeting dyes (Nile red and BODIPY 493/503), the authors revealed that the targeting ability of the CDs for LD can be attributed to their high lipophilicity. Other subsequent investigations also proved this lipophilicity targeting mechanism by using CDs prepared from 3-dimethylaminophenol,⁵⁹ 2,6-dibromo naphthalene dianhydride in ethylene glycol,⁹⁸ mixture of 1,2-diamino-4-fluorobenzene and thiourea in N,N-dimethylformamide,⁵⁸ and mixture of o-phenylenediamine and thiourea in N,N-dimethylformamide.⁹⁹ Gao et al. demonstrated the formation of a D–π–A structure via the Schiff base reaction between phenylenediamine and N,N-dimethylformamide, which are important precursors for the formation of LD-targeting CDs.⁵⁸ They also suggested that the lipophilic CH₃ group on the CDs played a vital role in determining the location of the as-prepared CDs in LD. By using the LD-targeting CDs, they observed the LD coalescence dynamics (Fig. 3C).⁹⁹

In contrast, fewer intrinsic ER-targeting CDs have been reported. In our previous investigations, two types of ER-targeting CDs were obtained via the hydrothermal treatment of mixtures of o-phenylenediamine and lysine/urea.^49,100 The co-localization studies proved the high specificity of the CDs towards ER (Fig. 3D).¹⁰⁰ Wu et al.⁵⁰ synthesized CDs via a facile one-pot hydrothermal method without additional modification, which possessed a positive charge, lipophilic surface, and boric acid groups, which endowed the CDs with two-stage cascade recognition ability for the endoplasmic reticulum. Wu¹⁰¹ obtained CDsomes_memvia the liquid–liquid extraction in butanol/water of the crude products from heating triolein at 220 °C for 3 days. The CDsomes_mem formed liposome-like structures and were distributed in the ER and other membrane systems due to their lipophilicity.

2.1.2 CDs generated from specific precursors. As elucidated above, numerous have demonstrated that organelle-targeting CDs can be achieved by treating unspecific precursors. However, the organelle-targeting results are not always reliable. For example, treating citric acid and N,N-dimethylaniline at 160 °C for 10 h produced CDs targeting lysosomes,¹⁰² whereas treating the same precursors under the identical conditions produced CDs targeting mitochondria and nucleolus in another study.⁹⁵ This inconsistent organelle-targeting can also be found for CDs produced from citric acid and urea.^11,26 Thus, due to the fact that CDs inherit their properties from their precursors, the preparation of CDs using precursors with specific affinity to certain organelles provides organelle-targeting results that are more reliable.

A typical example of this strategy is the generation of Golgi-apparatus-targeting CDs. According to previous reviews, the widely recognized targeting groups for the Golgi apparatus include norrisolide, phenyl sulfonamide, L-cysteine, kapakahines, bovine serum albumin-dyes complex, 7-aminoquinolines, and sphingolipids.¹⁰³ Huang's group⁵¹ reported the preparation of Golgi-targeting CDs from citrate acid and L-cysteine and demonstrated that L-cysteine remaining on the surface of the CDs combined with the sulfhydryl receptor site of the Golgi apparatus, which was the origin of their specificity towards the Golgi apparatus (Fig. 4A). They also achieved the precise delivery of ricin A-chain using the as-prepared Golgi-targeting CDs.⁵⁵ Penicillamine contains a similar structure to L-cysteine, and CDs prepared from D/L-penicillamine and citrate acid targeted the Golgi apparatus specifically.⁵³ Benzene sulfonamide has a selective affinity for cyclooxygenase-2 (COX-2), which is one of the signature proteins of the Golgi apparatus.¹⁰³ Thus, CDs generated from benzene sulfonamide and p-phenylenediamine could be used for the selective imaging of the Golgi apparatus via the specific affinity for COX-2.⁵⁴

Fig. 4 (A) Synthetic route of L-cysteine-rich chiral carbon quantum dots (LC-CQDs) and the Golgi imaging results, reprinted with permission from ref. 51, Copyright 2017, The Royal Society of Chemistry. (B) Schematic illustration of the preparation of CD-MB with boronic acid groups on its surface and its ER-targeting ability via a two-stage cascade ER recognition process, reprinted with permission from ref. 50, Copyright 2022, the American Chemical Society. (C) Illustration of preparation of lysosome-targeting CDs from citric acid and N,N-dimethylaniline, reprinted with permission from ref. 102, Copyright 2020, The Royal Society of Chemistry. (D) Schematic presentation of mitochondrial-targeting CDs from TPP, reprinted with permission from ref. 106, Copyright 2021, John Wiley and Sons. (E) Retrosynthesis of mitochondria-targeting CDs and mitophagy tracking, reprinted with permission from ref. 38, Copyright 2019, the American Chemical Society.

Another example illustrating this strategy is the dynamic visualization of endoplasmic reticulum stress in living cells using ER-targeting CDs prepared from 4-mercaptophenylboronic acid and ethylenediamine (Fig. 4B).⁵⁰ Boric acid groups can selectively connect with the o-dihydroxy group of mannose in the ER cavity, and thereby the as-prepared CDs achieved specific targeting of the ER. It should be noted that the positively charged and lipophilic surface of the CDs facilitated their accumulation in the ER, which also played an indispensable role.

In the case of lysosomes, the typical targeting groups are morpholine and N,N-dimethyl groups.¹⁰⁴ Li's group selected N,N-dimethylaniline and citric acid as precursors to prepare CDs and successfully obtained intrinsic lysosomal-targeting CDs with ultra-stability for long-term imaging (Fig. 4C).¹⁰² The as-prepared CDs localized in lysosomes in different cell lines such as HepG2, HL-7702, and 3T3 cells. By employing the photo-stability and long-term co-localization performance of the CDs, they achieved the tracking of lysosome movements under the stimulation of chloroquine.

Thiazole orange is a specific DNA/RNA-targeting dye. Yin et al. prepared nucleolus-targeting CDs by heating 2,4-dihydroxybenzaldehyde and 2,3-dimethylbenzothiazole iodide in a microwave reactor, in which the precursors contained thiazole groups.¹⁰⁵ The CDs showed selective affinity towards RNA, and thus localized in the nucleolus specifically. Besides, the small particle size and polarity response behaviour of the CDs realized fast nucleolus imaging effectively.

The use of triphenylphosphonium (TPP) as a precursor can ensure the achievement of mitochondrion-targeting CDs in most cases. Rajendran et al. employed a simple one-step carbonization method using TPP and PEG-di-NH₂ as carbon precursors and surface passivation reagent, respectively (Fig. 4D).¹⁰⁶ The TPP molecules carbonized and formed the CDs, while the amine groups of PEG-di-NH₂ attached or absorbed on the surface of the CDs to endow them with water dispersibility and enhance their fluorescence. Incubation with prostate cancer (PC-3), colon cancer (HCT116), neuroblastoma (SHSY5Y), and cervical cancer (HeLa) cells confirmed the mitochondrion-targeting ability of the CDs. The TPP moieties remained on the surface of the CDs, facilitating the labeling of mitochondria. Furthermore, the polarity and high lipophilicity of the residual TPP imparted excellent penetration in the mitochondrial intermembrane potential barrier.

Notably, Li's group modified the concept of retrosynthesis from organic chemistry to fabricate mitochondrion-specific CDs.³⁷ In their design of the synthetic route, citric acid and m-aminophenol formed a rhodamine structure, which served as both the mitochondria-targeting unit and luminescent center. They prepared mitochondria-tracking CDs (MitoTCD) from citric acid and m-aminophenol through microwave irradiation. By changing the substituent groups of m-aminophenol, several MitoTCDs with different fluorescence emissions and quantum yields were achieved. Employing the same strategy, they used citric acid and 8-hydroxyjulolidine to synthesize rhodamine-like fluorophores, which were further polymerized and carbonized to form another type of mitochondrion-specific CDs (Fig. 4E).³⁸ Although the organelle-targeting CDs prepared from specific precursors provide results that are more reliable, these studies are much less than that generated by unspecific precursors, which may hold great potential for developing organelle-targeting CDs.

2.2 Post modification with specific targeting species

Several molecules or groups are widely recognized as organelle-targeting species. For instance, morpholine is a targeting molecule for lysosomes and triphenyl phosphine is the most used group to construct mitochondrion-targeting probes.² Thus, post modification with these specific targeting species endows CDs specific targeting ability towards certain organelles. This post-modification strategy seems to provide the most reliable organelle-targeting results because the specific targeting species exist without any doubt.

2.2.1 Lysosome-targeting CDs. Xiang et al. reported the two-step post-modification of CDs to generate a lysosome-targeting nano-platform for the delivery of nitric oxide and photothermal therapy.⁴⁰ They prepared CDs decorated with carboxyl groups, followed by the first post-modification with ethylenediamine and second post-modification with morpholine-functionalized Ru complex and folic acid via covalent coupling. The distribution of the functionalized CDs matched well with that of Lyso-Tracker Red in HeLa cells with a Pearson correlation coefficient of 0.93. Meanwhile, in the contrast experiment in which the CDs were modified with the Ru complex without morpholine groups, they were randomly distributed in the cytoplasm and the Pearson correlation coefficient was 0.50. Subsequently, the delivery of nitric oxide and photothermal therapy of cancer cells were realized using the lysosome-targeting nano-platform.

He et al. simplified the procedures to prepare lysosome-targeting CDs.⁴¹ Initially, they prepared amino group-functionalized CDs via a one-pot method by heating a mixture of citrate acid and ethylenediamine rather than modifying carboxylated CDs with ethylenediamine. Subsequently, morpholine was covalently modified on the surface of the CDs to endow them with lysosome-targeting ability. Combining the fluorescence response of the 1,8-naphthalimide derivative to H⁺, the pH variation in the lysosome was tracked via fluorescence imaging.

In another investigation, Wu et al. prepared two types of lysosome-targeting CDs, i.e., CDs-MP and CDs-PEI-ML (Fig. 5A).⁴² CDs-MP was prepared by modifying aminating morpholine on the surface of carboxylic CDs, which were synthesized via the hydrothermal treatment of citrate acid. Alternatively, CDs-PEI-ML was prepared by modifying carboxylic morpholine on the surface of aminated CDs, which were synthesized via the hydrothermal treatment of a mixture of citrate acid and PEI. Both CDs-MP and CDs-PEI-ML targeted lysosomes with high specificity, high photostability, and low cytotoxicity. However, CDs-PEI-ML showed a 4-fold higher fluorescence intensity than CDs-MP, which indicates that CDs-PEI-ML is a more suitable fluorescent probe for lysosome imaging.

Fig. 5 (A) Illustration of preparation of lysosome-targeting CDs via modification with 4-(2-aminoethyl) morpholine, reprinted with permission from ref. 42, Copyright 2017, the American Chemical Society. (B) Design of lysosome-targeting ratiometric probe based on CDs, reprinted with permission from ref. 45, Copyright 2019, The Royal Society of Chemistry. (C) Illustration of the possible process for endocytosis and localization of NLS-CD complex in the nucleus, reprinted with permission from ref. 24, Copyright 2015, The Royal Society of Chemistry. (D) Presentation of the synthesis of mitochondrial-targeting CDs via modification with TPP and the subcellular localization of TPP-CDs and non-targeting CDs colocalized with MitoTracker Deep Red, reprinted with permission from ref. 65, Copyright 2014, The Royal Society of Chemistry.

Zhang et al. synthesized nanohybrid Ru1@CDs by modifying an Ru(II) complex (Ru1) on CDs.⁴³ Initially, they prepared intrinsic lysosome-targeting Ru1 and CDs, and then combined them via a simple condensation reaction using EDC and NHS as coupling agents. According to the colocalization experiments of Ru1@CDs, CDs, or Ru1 with commercial lysosome-targeting dyes (LTDR), the colocalization coefficients of Ru1@CDs, CDs, and Ru1 with LTDR were 0.88, 0.86, and 0.85, respectively. This demonstrated that Ru1@CDs, CDs, and Ru1 can specifically image lysosome. However, the photodynamic therapy results indicated that Ru1@CDs possessed higher potency of being an efficient agent for PDT treatment of cancer.

Chen et al. reported the synthesis of naphthalimide derivative-functionalized CDs (CDs-ND) for imaging formaldehyde in the lysosome (Fig. 5B).⁴⁵ They prepared the CDs by heating methionine and citric acid and obtained CDs-ND by covalently modifying the naphthalimide derivative on CDs. The colocalization experiment of CDs-ND with Lyso-Tracker Deep Red demonstrated the excellent lysosome-targeting ability of CDs-ND with a high Pearson colocalization coefficient of 0.93. Alternatively, CDs and ND only exhibited low Pearson colocalization coefficients of 0.46 and 0.56, respectively. The confocal laser scanning microscopy imaging results showed that the fluorescence signal of CDs-ND in HeLa cells responded to the content of formaldehyde in the lysosome, indicating the that CDs-ND is a favorable probe for monitoring the endogenous formaldehyde in living systems.

2.2.2 Nucleus- or nucleolus-targeting targeting. Besides the most important genetic molecules DNA and RNA in the nucleus, there are also active biomolecules in the nuclear pore complex (NPC), which may provide interaction sites for nucleus-targeting probes.

Yang proposed a nuclear localization signal (NLS) peptide-decorated CD conjugate to achieve targeted nucleus staining (Fig. 5C).²⁴ Initially, CD@PEG was prepared via the hydrothermal treatment of a mixture of PEG-2000, citrate acid and ethylenediamine at 160 °C for 8 h. Subsequently, NLS (PKKKRKVG) was covalently conjugated to CDs through the EDC/NHS coupling reaction to obtain the final NLS-CDs. After entering the cells, NLS-CDs combined with importin and formed an NLS-CDs-importin complex, and this complex passed through the nuclear pore to localize in the cell nucleus. Based on this study, they modified NLS-CDs with 4-hydrazinobenzoic acid (HBA) and conjugated DOX with the CDs through a hydrazone bond. The obtained conjugates delivered the anti-cancer drug DOX to the nucleus and killed cancer cells, which show potential as a promising nano-drug with enhanced therapeutic efficiency.¹⁰⁷

Tan et al. reported another example in which CDs were functionalized with a peptide to achieve cell nucleus-targeted imaging.²⁰ Initially, they synthesized CDs by heating a mixture of bovine serum albumin and formic acid, and then functionalized the CDs with the transactivator of transcription (TAT) peptide (GRKKRRQRRRPQ) to obtain TAT-CDs. The modification with the TAT peptide could overcome the lipophilic barrier of the cellular membrane and deliver the CDs into the cellular nucleus.

Small organic molecules can also be used to modify CDs and endow them with nucleus-targeting features. Barbosa et al. modified carboxylated CDs with ethylenediamine using thionyl chloride.¹⁷ The modified CDs are likely to have an affinity with ribosomal components, enabling the CDs to target the nucleus in the MCF-7 (breast cancer cells), MDA-MB-231 (breast cancer cells), Caco-2 (colorectal cancer cells) and DU145 (prostate cancer cells) cell lines.

Han et al. reported the synthesis of cationic carbon quantum dots that can distinguish nucleic acid structures and image the nucleus specifically.²¹ The carbon quantum dots were prepared via the oxidization of conductive carbon nanoparticles in refluxing HNO₃, acylation with p-phenylenediamine (pda), and chemical linkage with 4-carboxybutyl triphenylphosphonium (PPh³⁺) bromide. The carbon quantum dots could be inserted into the grooves of dsDNA and the fluorescence was enhanced. In contrast, the fluorescence emission was bathochromically shifted and a new red fluorescence peak centered at around 620 nm appeared when the carbon quantum dots were mixed with ssRNA. This different interaction endowed the carbon quantum dots capability to discriminate DNA and RNA in nuclei and nucleoli by green and red fluorescence easily, respectively. Moreover, long-term imaging of chromatin and nucleoli during cell division and the growth of Caenorhabditis elegans (C. elegans) was also achieved.

Wu et al. reported the modification of carbon nanodots with catechol–borane moieties for the nucleus-targeted delivery of doxorubicin.¹⁰⁸ The carbon nanodots were prepared by linking dopamine and 3-aminophenyl boronic acid with carboxylic CDs, which were denoted as p(CAT₂-CD-BA₁). The boronic acid on the outer shell of p(CAT₂-CD-BA₁) can bind to the glucosamine present in nucleoporin 62 (NUP62), which is the only glycoprotein existing in the inner ring of the NPC, endowing the CDs with nucleus-targeting ability. The authors also demonstrated that too much boronic acid modified on CDs over-interacted with the NUP62 gate and blocked the NPC channel.

2.2.3 Mitochondria-targeting CDs. To obtain mitochondria-targeting CDs, mitochondrially localized peptides and lipophilic/positive groups such as triphenylphosphonium (TPP) are the most frequently used targeting species (Fig. 5D).^65,109,110

Xu et al. conjugated a triphenylphosphonium (TPP) moiety and a photo-responsive NO donor on CDs to construct a mitochondria-targeted and NO-releasing nano-platform for cancer therapy.¹¹⁰ After cellular internalization, the nano-platform was distributed in the mitochondria and released NO, which caused high cytotoxicity towards cancer cells by specifically damaging their mitochondria. Wu et al. constructed a mitochondria-targeting nanoprobe by conjugating TPP on CDs obtained using o-phenylenediamine.²⁹ This nanoprobe was sensitive to peroxynitrite and its fluorescence decayed via the photoinduced electron transfer (PET) mechanism with a limit of detection of 13.5 nM. Cellular experiments demonstrated that this nanoprobe provides a practical tool for imaging peroxynitrite in mitochondria. Zhang et al. prepared a nano micelle via the assembly of TPP-modified D-α-tocopheryl polyethylene glycol succinate (TPGS) and 1-hexadecylamine-capped CDs.³⁴ The nano micelle delivered DOX to mitochondria, which overcame the multidrug resistance of cells and enhanced the treatment effect. Gong et al. developed a mitochondrial oxidative stress amplifier by modifying CDs supported by atomically dispersed gold with mitochondrion-targeting triphenylphosphine (TPP) and ROS-generating cinnamaldehyde (CA).¹¹¹ Upon mitochondria localization guided by TPP, the dispersed gold depleted mitochondrial glutathione, and thus amplified the reactive oxygen species damage caused by CA.

Shen et al. developed mitochondria-targeting supra-CDs via the modification of mitochondria-targeting peptides on CDs prepared by heating citric acid and dicyandiamide.¹¹² The mitochondria-targeting peptide (MLALLGWWWFFSRKKC) was conjugated with CDs covalently and endowed the CDs with mitochondria-targeting ability, which enhanced the photothermal therapy effect on cancer cells.

2.3 Uptake pathway of CDs

The endocytosis pathway of nanoparticles plays a significant role in their transport process and final subcellular locations. For example, the clathrin-mediated cellular uptake pathway usually delivers nanoparticles into the endosome, and then transports them to the lysosome, while the caveolae- or lipid-raft-mediated uptake pathway usually transports the nanoparticles to the endoplasmic reticulum or Golgi apparatus.¹¹³

Zhou et al. pioneered the investigation of the uptake pathway of carbon dots in neural cells (Fig. 6A).¹¹⁴ They demonstrated the involvement of passive diffusion, caveolae-mediated, and clathrin-mediated pathways, and revealed the distribution of CDs mainly within endo–lysosomal structures, Golgi apparatus, and nucleus, respectively. They also noticed that a small number of CDs was dispersed in the ER and mitochondria. They addressed the decisive role of the ultrasmall size (1–5 nm) and positive charge (ca. +2.3 mV) of CDs on their uptake pathway.

Fig. 6 (A) Schematic illustration of the uptake, intracellular trafficking and exocytosis of CDs in PC12 and RSC96 cells, reprinted with permission from ref. 114, Copyright 2014, The Royal Society of Chemistry. (B) Graphical description of the preparation, cellular endocytosis and localization in LoVo cells of CDs with different chemistry, reprinted with permission from ref. 47, Copyright 2021, the American Chemical Society. (C) Endocytosis pathway for CNPs in SH-SY5Y cells and a differentiated neuron, reprinted with permission from ref. 119, Copyright 2023, The Royal Society of Chemistry.

We discovered that the hydrophilicity/hydrophobicity and surface chemistry of CDs have significant effects on their uptake pathway and the final subcellular distribution. Hydrophilic CDs with surface amino groups could be internalized by cells mainly via clathrin-mediated endocytosis and located within the lysosome, while hydrophobic CDs decorated with laurylamine or short alkyl groups were internalized via caveolae-mediated endocytosis and membrane-penetrating passive diffusion and distributed in the endoplasmic reticulum (ER).^11,39 In a subsequent study, we designed four types of CDs with different surface chemistry and hydrophobicity, investigated their uptake pathway, and demonstrated their respective organelle-targeting feature (Fig. 6B).⁴⁷ In this study, lipophilic ECDs and NCDs decorated with CH₃ and C–O–C groups entered cells via a passive manner and localized in the ER and nucleus, amphiphilic GCDs with CH₃, C–O–C and NH₂ groups targeted the Golgi apparatus via caveolin-mediated endocytosis, while hydrophilic LCDs with CH₃, NH₂, and COOH preferred clathrin-mediated endocytosis and were distributed in the lysosome. These investigations proved the feasibility of organelle targeting by the design of surface chemistry and utilization of uptake pathways.

However, it should be noted that the uptake pathway of CDs by cells does not uniquely correspond to their organelles. For example, a combination of macropinocytosis-, clathrin-, caveolae-, and/or lipid raft-mediated pathways was identified in the internalization process of hydrophilic CDs that target the nucleolus.²³ Besides, the lipid raft- and clathrin-mediated endocytosis pathways not only delivered hydrophobic CDs to the ER and lysosomes, but also transported hydrophobic CDs to lipid droplets.^56,60 Nevertheless, a universal principle on the surface chemistry and uptake pathway by cells can be safely concluded, i.e., hydrophilic CDs with –NH₂, –OH, and –COOH groups are usually internalized via the clathrin-mediated pathway, hydrophobic CDs with alkyl/phenazine groups are usually uptake via the caveolae-, lipid raft-mediated pathway or direct passive diffusion, while amphiphilic CDs can be internalized via both the clathrin- and caveolae-mediated pathways.^114–117 A summary of the surface chemistry, uptake pathways, and subcellular locations of several organelle-targeting CDs is presented in Table 1.

Table 1 Summary of the surface groups, uptake pathway and subcellular location of several organelle-targeting CDs

Precursors	Surface groups	Uptake pathway	Subcellular location	Ref.
Citric acid and urea	–NH2 (ACDs), laurylamine (LCDs)	Clathrin-mediated (ACDs), clathrin- and lipid raft-mediated pathway (LCDs)	Lysosome (ACDs), endoplasmic reticulum (LCDs)	11
m-Phenylenediamine and L-cysteine	–OH, –NH2, –COOH, and C–S	Macropinocytosis-, clathrin-, caveolae-, and/or lipid raft-mediated pathways	Nucleolus	23
o-Phenylenediamine and lysine	CH3 (ECDs), C–O–C(NCDs), CH3, C–O–C and NH2 (GCDs), CH3, NH2 and COOH (LCDs)	Passive manner (ECDs, NCDs), caveolin-mediated pathway (GCDs), clathrin-mediated pathway (LCDs)	Endoplasmic reticulum (ECDs), nucleus (NCDs), Golgi apparatus (GCDs), lysosome (LCDs)	47
o-Phenylenediamine and urine	NH2, alkyl groups	Lipid raft-mediated and caveolin-mediated pathway	Endoplasmic reticulum (ECDs)	49
o-Phenylenediamine	Primary amine groups, phenazine structure	Lipid raft- and clathrin -mediated endocytosis	Lipid droplets	56
3-Aminophenylboro-nic acid	–OH, B-OH, NH2	Lipid raft-mediated endocytosis	Lipid droplets	60
Citric acid and ethylenediamine	–OH, NH2	Caveolae- and clathrin-mediated pathways, passive diffusion	Nucleus, endo–lysosomal structures and Golgi apparatus	114
Grape seed extract	–OH, –COOH	Caveolae-and clathrin-mediated endocytosis	Nucleus	115
o-Phenylenediamine and phenylalanine	Benzene ring, nitrogen heterocyclic ring, amino and hydroxyl	Passive diffusion	Endoplasmic reticulum	116
Rose bengal and DL-cysteine	N–H, S–H	Clathrin- and lipid raft-mediated endocytosis	Endoplasmic reticulum	117
Commercially acquired CDs	3-Ethoxypropylamine, oligomeric polyethylenimine	Clathrin- and caveolae-mediated and pathways, macropinocytosis	Lysosome	118
p-Phenylenediamine and diphenyl ether	NH2	Clathrin-mediated	Lysosome	119

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Recently, in-depth research on the cellular pathway of CDs has emerged. Yan et al. investigated the effect of the surface functionalities of CDs on their endocytosis in different cell cycle phases.¹¹⁸ They used 3-ethoxypropylamine-functionalized CDs (EPA-CDots) and oligomeric polyethylenimine-functionalized (PEI-CDots) as models. The results showed that the HeLa cells internalized overall slightly more PEI-CDots. In the presence of serum, EPA-CDots were internalized mainly via clathrin-mediated endocytosis and caveolae-mediated endocytosis, which functioned only in the S phases. In the case of PEI-CDots, caveolae-mediated internalization and macropinocytosis were the major pathways. However, regardless of the endocytosis pathway, both CDs were localized in the lysosomes. Besides, Hivare et al. investigated the endocytic pathways of CDs in neuronal cells at different stages of differentiation (Fig. 6C).¹¹⁹ They employed red fluorescent CDs synthesized through the reflux reaction of p-phenylenediamine with diphenyl ether, which possessed an aromatic ring of carbon and C–NH₂ groups. When incubated with SH-SY5Y-derived neuroblastoma neurons, the CDs were preferably taken up via clathrin-mediated endocytosis and targeted lysosomes in undifferentiated cells and differentiated cells at different stages. These investigations provide an in-depth understanding of the cellular uptake of CDs, but the targeting by the CDs was limited to lysosomes. Thus, the uptake of other organelle-targeting CDs by different cells or cells at different cell cycle phases needs more attention in the future.

3. Challenges and perspectives

3.1 Rational design and precise preparation of organelle-targeting CDs

3.1.1 Improving the reproducibility and specificity of organelle targeting. To date, there have been abundant investigations on the development of different types of organelle-targeting CDs and subcellular imaging of organelle components or physiological parameters such as pH value and polarity.^{11,36,46,71,100,109} However, the rational design and precise preparation of organelle-targeting CDs are still challenging.

In the case of intrinsic organelle-targeting CDs, the most significant issue is the reproducibility of their targeting feature. As elucidated above, there are examples on the synthesis of CDs using identical precursors under identical conditions, but CDs targeting different organelles were produced.^11,26,95,102 In addition, many CDs are decorated with similar surface chemistry but target different organelles.^13,69,72,96 Moreover, there are also investigations in which CDs prepared from different precursors and possessing different surface chemistry/charge, hydrophilicity, and size could target the same organelle.^{28,46,73,90,120} Thus, the organelle selectivity of intrinsic organelle-targeting CDs needs to be improved (Fig. 7A).^71,121,122 These issues remind us that knowledge on CDs is far from sufficient to understand and explain these phenomena.

Fig. 7 (A) Illustration of the synthesis of CDs that target mitochondria and lysosome simultaneously, reprinted with permission from ref. 71, Copyright 2020 Elsevier Ltd. (B) Schematic presentation of column separation of CDs, reprinted with permission from ref. 133, copyright 2019, The Royal Society of Chemistry. (C) Illustration of regulation of morphology of CDs and the improvement in their ER-targeting specificity, reprinted with permission from ref. 140, Copyright 2023, John Wiley and Sons.

CDs do not possess definite structures and exact molecular weights. However, it is clear that CDs are usually composed of carbon, oxygen, and hydrogen elements, and some are doped with nitrogen, phosphorus, sulphur, or other elements.^10,123 Another consensus is that most CDs present a core–shell-like structure, and the composition and structure of their carbon core and surface group shell can be regulated by changing the precursors and reaction conditions.^124,125 For a long time, researchers have devoted their efforts to producing various organelle-targeting CDs by regulating the precursors, optimizing the reaction conditions, and modifying CDs with specific moieties. However, there are various types of reactions between the precursors and these complex reactions lead to the production of multiple mixtures including carbon dots, unknown fluorescent molecules, or their combination, as well as other impurities and by-products.^{126,127–130} Thus, this complex composition may be the origin of the poor reproducibility of CDs, which make the rational design and precise preparation of CDs a difficult task. Considering this, the intrinsic targeting ability of one-pot prepared CDs seems to be a trial-error strategy to a certain degree. Notably, retrosynthesis methods in which the precursors were designed and the reactions between them were controlled made the intrinsic targeting CDs more precise and reliable.^37,38 However, the retrosynthesis method is limited to mitochondria and particular precursors and suitable synthesis methods have not been explored in the synthesis of CDs targeting other organelles.

Rigorous purification by a combination of filtration, centrifugation, and dialysis with chromatography or gel electrophoresis can remove the by-products and obtain purified carbon dots with more reliable results.^{120,131–137} A typical example is the achievement of four different CDs targeting the ER, nucleus, Golgi apparatus, and lysosome from the purification of CDs using silica column chromatography and Sephadex.⁴⁷ Separation of the one-pot synthesized CDs also provided evidence for the structure analysis and surface-state-controlled luminescence mechanism (Fig. 7B).^133,135 To date, rigorous purification has been ranked as the most effective protocol to ensure the reproducibility of organelle-targeting CDs, which deserves to be popularized in the future.

In addition, new artificial intelligence methods such as machine learning may provide effective tools for the rational design of organelle-targeting CDs given that they assisted in the synthesis of CDs with predictable emission color and wavelength.^138,139

In comparison with the intrinsic organelle-targeting strategy, the post-modification strategy avoids controversy through the use of specific targeting moieties, which helps to avoid the “trial-error” trap. However, specific targeting ligands change target sometimes. For example, the mitochondria-targeting ligand TPP endows CDs with a highly positive surface and facilitates the nucleoli imaging.²¹ Thus, the question arises whether can we use TPP to prepare nucleoli-targeting CDs? Besides, the post-modification strategy suffers from a lack of specific targeting moieties.

In summary, the reproducibility and specificity of the organelle-targeting ability of CDs need to be improved. Recently, Dr. E et al. reported an anion-directed strategy to control the morphology of fluorescent carbon nanoparticles from spherical to rod-shaped (Fig. 7C).¹⁴⁰ All four types of carbon nanoparticles targeted the endoplasmic reticulum (ER), but their ER-targeting specificity was enhanced as their shape changed from spherical to higher-AR rods, accompanied by an increase in the Pearson correlation coefficient from 0.71 to 0.90. This investigation not only presented a new strategy to regulate the morphology of carbon dots, but also demonstrated that the morphology of CDs may be another breakthrough point to enhance their organelle-targeting specificity.

3.1.2 Development of CDs targeting more subcellular structures. Subcellular imaging is a powerful tool to visualize the evolution of organelle structures, reveal the interactions between organelles, and analyse the variation in the content of biomolecules.¹⁴¹ Thus, to realize this goal, comprehensive observations about the intracellular structures are of great importance, which demands reliable imaging results from all subcellular structures. However, the reported CDs to date exhibit limited targeting of the organelles in the nucleus/nucleus, mitochondria, lysosome, Golgi apparatus, lipid droplets, and endoplasmic reticulum. There are rare reports about CDs targeting the cytoskeleton, centrosomes, vesicles, lipid membrane system, peroxisome, etc.⁶³ In addition, there is also demand that CDs discriminate the evolution and differences of a particular organelle at different statuses, including CDs tracking the delivery and fusion of endosomes with lysosomes, discriminating the granular endoplasmic reticulum and smooth endoplasmic reticulum, and visualizing the interaction between different organelles. In summary, new methods or strategies need to be explored for the further investigation of the organelle/structures that have been imaged by CDs, and new CDs need to be developed for organelle/structures that have not been imaged by CDs. In this case, give that organic molecular probes have been proven to have potential in multiple organelle-targeting imaging, the design and synthesis of organic fluorescent probes may provide some inspirations.^2,142

3.2 The organelle-targeting mechanism

3.2.1 Diverse explanations of the targeting mechanism need consistency. The theoretical investigation of the mechanism is vital for further design and preparation of organelle-targeting CDs. However, there are diverse explanations for the organelle-targeting mechanism of CDs, especially for the intrinsic organelle-targeting strategy.

Usually, the targeting ability of CDs is attributed to their surface chemistry. However, the explanations for the targeting mechanism of functional groups are hardly consistent.¹⁴³ For example, the weak alkaline or acidotropic effect of amino groups on the CDs was the most noted mechanism for lysosome-targeting CDs.^{44,69,144,145} However, the targeting ability of amine groups for lysosomes is not specific and amino-group-decorated CDs not only target lysosome, but also target the nucleus/nucleus^77,79,146 and mitochondria,^32,33,36 or target both lysosomes and mitochondria simultaneously.⁷¹ When CDs were observed to be located in the nucleus or mitochondria, the targeting mechanism was usually attributed to their surface positive charge or the combination of positive charge with appropriate lipophilicity.^{36,71,77,79,147} Although these proposed mechanisms can explain the experimental observation, there are still queries. For example, both the weak alkaline/acidotropic effect and the positive surface charges originated from the amino groups on the surface of CDs, but how can the amino groups selectively display different properties and locate in different organelles? If we need a type of nuclear-targeting CDs, should we consider amino group-decorated CDs? Moreover, there are investigations proving that CDs with negative surface charges can also target the nucleolus^85,86 and mitochondria,^148,149 which is inconsistent with the commonly recognized charges “repel and attract” principle. In summary, the explanations for the mechanism of the organelle-targeting ability of CDs are contradictory. Thus, new explanations need to be proposed.

There are multiple factors affecting the subcellular distribution of CDs, and although some of these factors have been investigated, some have been rarely noticed. One of the investigated factors is the cellular uptake pathway of CDs. When this factor was considered, rational and satisfactory explanations were proposed to explain why different CDs shared similar surface groups or charges but targeted different organelles.^11,56,116 Besides, the uptake pathway of CDs was found to be dependent on their physical/chemical properties, such as surface chemistry, hydrophilicity, size, and charges.^11,47,118 A raw principle can be addressed on the relationship between the physical/chemical properties and uptake pathways. However, the uptake pathway strategy does not result in specific organelle targeting sometimes. This is may be due to the fact that one endocytosis pathway can transport probes to several different organelles and the CDs can be internalized via several pathways simultaneously.^{23,75,117,150,151}

In summary, reasonable targeting mechanisms have been proposed, especially when the uptake pathway was considered. However, there is still a long way to go to achieve consistent explanations for the targeting ability of CDs. To illustrate the targeting mechanism more clearly, we summarise the typical CDs targeting different organelles based on their raw materials and the targeting mechanism in Table 2.

Table 2 Summary of the raw materials and the targeting mechanism of typical organelle-targeting CDs

Organelle	CDs	Raw materials	Targeting mechanism	Ref.
Lysosome	ACDs	Citric acid monohydrate and urea	Clathrin-mediated endocytosis	11
	CDs	Citrate acid, ethylenediamine, and morpholine	Weak alkaline or acidotropic effect of morpholine groups	41
	CDs-PEI-ML	CA, PEI, and morpholine		42
	CDs	Benzoquinone and ethylenediamine	Alkaline –NH2 groups	44
	CDs	Dexamethasone and 1,2,4,5-tetraaminobenzene		46
	P-R CDs	Rose bengal and branched polyethyleneimine		70
Mitochondria	CDs	Mercaptosuccinic acid, ethylenediamine, and chitosan	Lipophilicity and positive charges	32
	s-GQDs	Perylene tetracarboxylic anhydride and polyethylenimine		36
	APTMS CDs	Glycerol and silane		33
	N,S-CNDs	α-Lipoic acid, citric acid, and urea	Electronic interaction	91
	C-dots-TPP	o-Phenylenediamine, TPP	lipophilicity and positive charges of TPP	29
	MitoCAT-g	Citric acid, TPP		111
	SCDs	Citric acid and dicyandiamide	mitochondria-targeting peptides	112
Nuclear/Nucleolus	QCDs	Tris and betaine	Positive charge	16
	N-CDots	Dopamine		19
	Ni-pPCDs	p-Phenylenediamine/Ni^2+		27
	TAT-CDs	Bovine serum albumin and formic acid	Specific affinity of peptides	20
	NLS-CDs	PEG-2000, CA, ethylenediamine, and NLS		24
	CDs	Ascorbic acid	Non-covalent interaction	28
	HBIE-CDs	m-Phenylenediamine and folic acid	Hydrogen bonding	86
	FCDs	Sodium hydroxide and polyethylene glycol	Protonation of oxygen groups	87
	C-CD/TiO2	Dopa-decyl	Zwitterionic surface	88
Golgi apparatus	LC-CQDs	Citrate acid and L-cysteine	Specific affinity of L-cysteine	51
	D/L-Pen-CDs	D/L-Penicillamine and citrate acid	Specific affinity of sulfonamide	53
	GTCDs	p-Phenylenediamine and benzene sulfonamide		54
Endoplasm reticulum	LCDs	Laurylamine, EDC·HCl and NHS	Caveolae-mediated endocytosis	11
	CD-MB	4-MPBA, 4-CPBA, and 4-MBA	Positive charge, lipophilic surface, and boric acid groups	50
Lipid droplets	CDs	3-Dimethylaminophenol	Lipophilicity, endocytosis pathway	59
	PA CDs	4-Piperidinoaniline		61
	CDs	2,6-Dibromo naphthalene dianhydride		98

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3.2.2 The intracellular transport process. When CDs are incubated with cells, the CDs first interact with the molecules on the cell membrane, and then internalized by the cells via different pathways, followed by intracellular transport, and finally localized in the subcellular organelles. Therefore, comprehensive investigations on the uptake pathway, intracellular transport process, and interaction of CDs with intracellular molecules will provide more evidences to determine the real organelle-targeting mechanism. The uptake pathways of CDs have been studied preliminary, but the influence of the intracellular transport on the organelle distribution of CDs has been seldom noticed.

As demonstrated by previous investigations, most nanoparticles are dominantly internalized via clathrin-mediated endocytosis and are trafficked to lysosomes.¹¹³ Thus, lysosome escape of nanoparticles is vital to determine their intracellular trafficking and final subcellular locations. It can be speculated that some CDs target lysosomes due to their failed lysosome escape rather than their real lysosome targeting ability. This can also help to explain why some amino-group-surfaced CDs are located in lysosomes but CDs possessing similar surface groups enter the nucleus or are attached to mitochondria.

In this case, the appropriate design of the surface chemistry of nanoprobes can enable endosome/lysosome escape.¹⁵² Post modification with the NLS peptide is an efficient way to help CDs escape from lysosomes and target the nucleus, as illustrated in section 2.2.2.²⁴ Besides, surface groups that are readily protonated in the lysosome can induce the entry of excess water and splitting of the lysosome, and subsequently the CDs can escape from the lysosome.¹⁵³ Besides, the lysosome escape of CDs is cell-line dependent. The design of the surface chemistry can also avoid non-clathrin-mediated endocytosis, bypass endosomal trafficking, and target different subcellular organelles.¹⁵² The preparation of CDs with amphiphilicity, lipophilicity, or hydrophobicity enables their caveolae/lipid raft-mediated endocytosis and passive diffusion, which makes CDs target mitochondria, endoplasmic reticulum, Golgi apparatus, and lipid drops.^{11,47,49,56,60,114–119}

The molecules on the cell membrane can be used to construct the surface chemistry of CDs. Nasrin reported the synthesis of folic acid fragment-decorated CDs, which were combined with a folate receptor and glycosyl-phosphatidyl-inositol (GPI) anchor to form a hybrid. After entering the cells, the hybrid was involved in endosomal transport and cleaved by lysosomal action by GPI. Subsequently, the CDs were released around the nucleus, where they penetrated the nucleus through nuclear pore complexes.¹⁵¹

Recently, the migration of CDs between organelles has been investigated. Guo et al. observed the migration of CDs between mitochondria and nucleolus when the mitochondria membrane potential (MMP) changed. The CDs migrated from the mitochondria to nucleolus when the MMP increased, and returned to the mitochondria when the MMP recovered.⁹⁵ Gao et al. tracked lipid droplet dynamics by using amphiphilic carbon dots and found that LDs with green fluorescence (emitted by CDs) gradually accumulated on the mitochondria during starvation.⁵⁸ Considering that organelle-organelle interactions are essential for maintaining intracellular homeostasis,¹⁵⁴ these special discoveries may initiate new insight into understanding the transport of matter between organelles.

It can be seen that intracellular transport investigations produced more results on how to enhance the lysosomal escape or avoid the clathrin-mediated pathway. However, the transport process after internalization via non-clathrin pathways needs further investigations to determine the real intracellular fate of CDs.

3.2.3 Interaction with intracellular molecules. Nanoparticles begin to interact with molecules on the outer surface of cell membrane and in the cytoplasm once they contact cells.¹⁵⁰ Thus, the interaction between CDs and specific intracellular molecules is the basic principle for understanding the targeting mechanism. If the active molecules playing vital roles during the internalization and transport process can be identified and the interaction between the CDs and the active molecules can be revealed, we can propose the organelle-targeting mechanism of CDs from a molecular perspective, which probably produces a targeting mechanism without contradictions.

The necessity to study the interactions between CDs and intracellular molecules has been demonstrated by several previous reports.

As is known, triphenylphosphonium (TPP) is a widely used reagent to endow nanoparticles with mitochondria-targeting feature and mitochondria-targeting CDs can be feasibly prepared by modification with TPP.^{23,34,110,111} The targeting ability is usually attributed to the electrostatic interaction between the highly positive surface of CDs induced by TPP and the highly negative surface of the membrane of mitochondria. However, the electrostatic interaction is not specific and the highly positive surface theoretically endows CDs interaction with the negative membranes of other organelles. Nevertheless, there are rare reports in which TPP-modified CDs targeted the endoplasmic reticulum, lysosome, Golgi apparatus, or lipid droplets. Therefore, we think it is better to reconsider the targeting mechanism of TPP-modified CDs by identifying the specific molecules functioning in the mitochondria-targeting process. Recently, an innovative study demonstrated the targeting imaging of nucleic acid structures rather than mitochondria by using TPP-modified CDs,²¹ in which the specific affinity of TPP towards DNA and RNA was evidenced solidly by experimental results. This study demonstrated that the interaction between CDs and DNA/RNA endowed TPP-CDs nucleus-targeting ability, which elucidated the targeting mechanism without any doubts. This example proved the significance of investigating the interactions between CDs and intracellular molecules.

Both DNA and RNA are polyanions and exhibit negative features in normal intracellular physiological circumstances. Theoretically, CDs with positive surfaces rather than negative surfaces target the nucleus easily due to their interaction with DNA or RNA.^{16,19,27,77–84} However, several investigations demonstrated that negatively charged CDs can also target the nucleus/nucleolus.^{22,23,28,85–87} Non-covalent interactions²⁸ and hydrogen bonding⁸⁷ with DNA/RNA were proposed to explain the targeting mechanism of CDs. Besides, the interaction between oxygen group-modified CDs and alkaline chromatin was also attributed to the reason for their nucleus-targeting ability.⁸⁸ Although CDs mostly exhibit unspecific interactions with intracellular molecules, which result in artifacts, the study on these interactions is beneficial for the elucidation of their targeting mechanism.

Nevertheless, the active molecules in the endoplasmic reticulum, lysosome, Golgi apparatus, lipid droplets, and other subcellular structures have not been revealed, which need more attention and may become a potential field of study. Besides, active biomolecules functioning in the endocytosis process and the intracellular transport can also provide evidence for the subcellular targeting of CDs. To carry out related investigations, knowledge of molecular biology and cell biology should be emphasized and related investigation methods should be introduced.

3.2.4 Super-resolution imaging techniques. Seeing is believing, and thus clear and bright cellular imaging results with high spatial resolution make it easy to explain the experimental results. Super-resolution imaging techniques have broken the diffraction resolution limit (200 nm) and imaged specific proteins in the lysosome and mitochondria, organelle dynamics, and rapid subcellular processes.^155–159 For example, Li et al. extended the resolution of structured illumination microscopy to 45–84 nanometres and imaged assemblies of clathrin and caveolin, as well as the dynamics of mitochondria, actin, and the Golgi apparatus.¹⁵⁷ Qiao et al. developed rationalized deep learning for structured illumination microscopy and lattice light sheet microscopy and achieved imaging of the rapid kinetics of motile cilia and nucleolar protein condensation.¹⁵⁹ Therefore, it is rational to speculate that super-resolution imaging techniques can help to reveal the uptake, intracellular transport, and final distribution of CDs more precisely.

Recently, super-resolution imaging of subcellular organelles using CDs as fluorescence probes produced interesting results. Hua et al. reported the super-resolution imaging of the nucleolus with a spatial resolution of 146–172 nm using red-emitting CDs, which presented a clearer nucleolus structure than conventional confocal imaging.²⁷ In another report, He et al. designed CDs integrated with fluorescent blinking domains and RNA-binding motifs, which achieved super-resolution imaging of the nucleolar ultrastructure. The authors used the CD-depicted nucleolar ultrastructure as a hallmark to distinguish different cell types accurately and identify subtle responses to various stressors (Fig. 8A).¹³⁴ In addition, by using stimulated emission depletion microscopy and endoplasmic reticulum (ER)-targeting CDs, Li observed the dynamic process of ER from loosely spaced tubes in a continuously dense network of tubules and sheets during cell division (Fig. 8B).¹¹⁶ They also found that the ER can contact with almost every other organelle.

Fig. 8 (A) Illustration of the preparation of ER-targeting CDs and confocal and STED images of ER, reprinted with permission of ref. 116, Copyright 2022, The Royal Chemistry Society. (B) Schematic illustration of the structures of RBP- and N-CDs and STORM images of nucleolus structure of different cell lines, reprinted with permission of ref. 134, Copyright 2021, the American Chemical Society.

However, although some remarkable progress has been made in the application of CDs in super-resolution imaging, there are still great limitations, such as the aggregation and nonspecific binding of CDs, which may lead to artifacts.¹⁶⁰ Besides, super-resolution imaging is expensive, which hinders its wide application. Therefore, designing the surface chemistry of CDs and developing new super-resolution imaging techniques to reduce the cost need more attentions. In summary, super-resolution microscopy can provide more details in organelle imaging with CDs as probes and help to understand the intracellular fate of CDs, but related investigations are still in their infancy.

4. Conclusions

In conclusion, we outlined the development of organelle-targeting investigations using CDs as probes by summarizing the organelle-targeting strategies, including the intrinsic targeting ability of one-pot CDs, post-modification with specific species, and uptake pathways of CDs. We also indicated the challenges in this field in which the reproducibility and specificity of organelle-targeting features and the targeting mechanism of CDs were emphasized. Potential solutions were proposed from the perspective of cells and organelles, in which the intracellular transport of CDs, their interaction with intracellular molecules, and super-resolution imaging techniques were highlighted. We believe that this review will provide useful insights to realize greater achievements in the field of organelle fluorescent imaging.

Author contributions

Quanxing Mao: conceptualization, funding acquisition, supervision, writing – original draft, writing – review & editing. Yujie Meng: resources, writing – original draft. Yuhang Feng: resources, writing – original draft. Hui Li: funding acquisition, supervision, writing – review & editing. Tianyi Ma: funding acquisition, project administration, supervision, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge the support from the National Natural Science Foundation of China (no. 21804062, 52071171, 52202248), Liaoning BaiQianWan Talents Program (LNBQW2018B0048), Shenyang Science and Technology Project (21-108-9-04), Key Research Project of Department of Education of Liaoning Province (LJKZZ20220015), Australian Research Council (ARC) through Future Fellowship (FT210100298, FT210100806), Discovery Project (DP220100603), Linkage Project (LP210100467, LP210200504, LP210200345, LP220100088), and Industrial Transformation Training Centre (IC180100005) schemes, and the Australian Government through the Cooperative Research Centres Projects (CRCPXIII000077).

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