WO2016133462A1

WO2016133462A1 - Conjugated polymer nanodots as long-term stem cell trackers

Info

Publication number: WO2016133462A1
Application number: PCT/SG2016/050081
Authority: WO
Inventors: Kai Li; Guorui JIN; Bin Liu
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2015-02-16
Filing date: 2016-02-16
Publication date: 2016-08-25
Anticipated expiration: 2017-08-16
Also published as: SG11201706685PA

Abstract

This invention relates to a method for tracking stem cells characterized in that the stems cells are labelled with at least one fluorescent conjugated polymer nanodot and the fluorescence of the labelled cells is detected. The fluorescent conjugated polymer nanodot comprises a conjugated polymer such as poly(9,9-dihexylfluorene-alt-2,1,3-benzoxadiazole) (PFBD) capped with an amphiphilic co-polymer such as DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-conjugated polyethylene glycol) and DSPE-PEG₂₀₀₀ maleimide. The method allows for the tracking of stem cells over long periods of time after transplantation exceeding 25 days. The stem cells are not compromised in their behaviours of proliferation, migration, differentiation and secretome by the labelling method of the invention. The method may be useful as a research tool in regenerative stem cell medicine. Corresponding stem cell tracking kits are also disclosed as well as stem cells labelled with the fluorescent conjugated polymer nanodots.

Description

Title of Invention: Conjugated polymer nanodots as long-term stem cell trackers Technical Field

The present invention relates to a method for tracking stem cells by use of conjugated polymer nanodots and the stem cells obtained with this method. The method may be used to reveal mechanisms in skin regeneration by measuring the fluorescence of transplanted cells.

Background Art

Stem cell-based therapies hold great promise in providing desirable solutions for diseases that cannot be effectively cured by conventional therapies. While there has been a rapid surge in clinical trials involving mesenchymal stem cells (MSCs), few treatments have been translated to humans due to contradictory results. One of the reasons for the conflicting results is the lack of effective long-term cell tracking approaches that are able to promote comprehensive understanding of the fate (e.g., survival, migration, differentiation and engraftment) of transplanted MSCs without impairing their intrinsic properties. For instance, the commercially well recognized standard of direct labelling probes (commercial product Qtracker®), have been reported to greatly diminish the MSC function upon labelling and failed to conclusively track cell location during in vivo bone regeneration study. It has been found that MSCs could contribute to wound repair by transdifferentiation into multiple skin cell types (including keratinocytes), while bioactive factors released by MSCs were found to make major contributions in skin regeneration through regulating the local cellular responses to injury. However, the exact behaviours of transplanted MSCs (e.g., engraftment and migration) during skin regeneration have not been comprehensively deciphered, due to lack of a facile and reliable long-term stem cell tracking system.

Organic cell trackers have been reported previously, but were only used on cancer cells.

Stem cell therapeutic models require a long-term performance of these trackers in the environment of stem cells. Extensive screening on animal models of skin regeneration would be needed and so far no long-term well performing trackers have been identified. A variety of stem cell behaviours (including viability, proliferation, migration, differentiation and paracrine signaling) after labelling must remain uninfluenced to develop a commercially usable tracker. The tracker must at the same time provide high photostability, high quantum yield and low cytotoxicity in the stem cell uses.

Commercial standard of direct labelling stem cell probes have been reported to greatly diminish the MSC function upon labelling and failed to conclusively track cell location during in vivo bone regeneration study. However, minimal perturbation to the functions of transplanted stem cells (proliferation, migration, differentiation and paracrine signalling) would be desirable.

There is therefore a need to provide a method for tracking stem cells, especially in skin regeneration uses, that overcomes or at least ameliorates, one or more of the disadvantages described above and shows a reliable racking ability over elongated times of transplanted stem cells.

Summary of Invention

According to the invention conjugated polymer (CP) nanodots as non-invasive fluorescent trackers with high brightness and low cytotoxicity for tracking of mesenchymal stem cells (MSCs) to reveal their in vivo behaviours have been found.

In an aspect, there is provided a method for tracking stem cells characterized in that the stems cells are labelled with at least one fluorescent conjugated polymer nanodot and the fluorescence of the labelled cells is detected.

Advantageously, the tracking by CP nanodots shows significantly better long-term tracking ability without compromising the features of MSCs in terms of proliferation, migration, differentiation and secretome compared with commercial standards. The labelling approach using CP nanodots makes it feasible to provide valuable insights into the regenerative capacities of therapeutic stem cells, especially those cells with difficulties in transfection to express fluorescent proteins or luciferase. According to the invention is possible to use fluorescence imaging of tissue sections from full- thickness skin wound-bearing mice transplanted with CP nanodot-labelled MSCs. The method allows toevaluate the effect of paracrine signalling of the MSCs residing in the regenerated dermis on skin regeneration. Using exogenous cell trackers the method further allows to clearly visualize whether the transplanted MSCs did or did not undergo epidermal differentiation by checking whether the cells only remain in the regenerated dermis without migration to the epidermis. It further allows an insight into the impact of nanodot internalization on cell differentiation from a biophysical point of view.

Further advantageously, the labelling approach using CP nanodots is feasible to provide valuable insights into the regenerative capacities of therapeutic stem cells, especially those cells with difficulties in transfection to express fluorescent proteins or luciferase. Further advantageously, the fluorescent CP nanodots have extremely high extinction coefficients and high quantum yield over long observation periods. The conjugated polymer based stem cell tracker is cost effective.

Advantageously, the method provides the use of exogenous labelling probes for long-term stem cell tracking to investigate the roles of transplanted stem cells in regenerative medicine. It allows the visualization transplanted MSCs and to investigate epidermal differentiation of them.

Advantageously, the fluorescent CP nanodot labelleing does not interfere with parallel bioluminescence measurements.

Advantageously, in an animal model of skin regeneration the use of the method according to the invention showed good performance through both in vitro and in vivo approaches.

In another aspect, there is provided a method that utilizes a conjugated polymer nanodot which comprise a conjugated polymer capped with an amphiphilic co-polymer.

Advantageously, the use of such nanodots is especially efficient in labelling the stems cells and allows for the tracking and visualization of stem cells over periods of about 1 to 28 days, or in certain cases even longer than 28 days. Advantageously, the nanodots to not have any effect on the stems cells development and are non-toxic.

In another aspect, there is provided a use the method of the invention for investigating the regeneration of skin wherein stem cells have been transplanted.

Advantageously, the observation according to the use can be done over long periods exceeding ten days without any significant loss of tracking ability.

In yet another aspect, there is provided a kit for stem cell tracking comprising a container with at least one fluorescent conjugated polymer nanodot together with other excipients for applying the nanodots to the cells or instructions on the use of the nanodots.

In a further aspect, there is provided a stem cell labelled with the fluorescent conjugated polymer nanodot.

Advantageously, the behaviour of such stem cells can be observed by visualisation over long terms after transplantation.

Definitions

The following words and terms used herein shall have the meaning indicated: The term "stem cell" refers to an undifferentiated cell of a multicellular organism which is capable of giving rise to indefinitely more cells of the same type, and from which certain other kinds of cell arise by differentiation.

The term "fluorescent conjugated polymer" as used herein refers to a polymer that displays fluorescent properties, including oligomers such as dimers, trimers etc. and copolymers, which are fully conjugated (i.e. are conjugated along the entire length of the polymer chain) or are partially conjugated (i.e. which include non- conjugated segments in addition to conjugated segments) . The conjugated polymers may have π-electron delocalized backbones.

The term "fluorescent conjugated polymer (nano)dot" used herein refers to a polymer (nano)dot that displays fluorescent properties and comprises one or more fluorescent conjugated polymers in the form of nanometer-scale localized structures.

The term "nanodot" as used herein refers to nanometer-scale localized structures. It preferably refers to particles possessing dimensions less than about 1000 nm.

The term "aprotic solvent" as used herein refers to a polar solvent which does not contain acidic hydrogen and does not act as a hydrogen bond donor.

The term "protic solvent" as used herein refers to a solvent that contains a dissociable H⁺ ion.

The term "labbelled with at least one fluorescent conjugated polymer nanodot" as used herein refers to the state of a cell that is made identifiable by attaching at least one fluorescent polymer nanodot to it. The term "fluorescence" refers to the emission of light by a substance that has absorbed light or other electromagnetic radiation.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Detailed Disclosure of Optional Embodiments

Exemplary, non-limiting embodiments of a polymer matrix will now be disclosed. There is provided a method for tracking stem cells characterized in that the stems cells are labelled with at least one fluorescent conjugated polymer nanodot and the fluorescence of the labelled cells is detected.

The nanodot may have dimensions in the range of about 5 to about 50 nm, or about 10 nm to about 500nm, or about 50 nm to about 300 nm, preferably about 30 nm to about 50 nm, about 30 nm to about 45 nm, 30 nm to about 40 nm, 30 nm to about 35 nm, 35 nm to about 50 nm, 40 nm to about 50 nm, or 45 nm to about 50 nm. The size of the nanodots may in average be about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. Preferably it has a particle size of about 5 to 50 nm, preferably 10 to 20 nm. A particle size of about 30 to 40 nm (hydrodynamic diameter) may be most preferred. The labelling of the stem cells may be achieved by incubation with the nanodots.

The fluorescence is usually detected at another wave length than the wave length of the absorbed light. The emitted fluorescence may a longer wavelength than the absorbed light, and therefore lower energy, than the absorbed radiation.

The stem cell may be a mesenchymal stem cell (MSC). MSCs are multipotent stromal cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), adipocytes (fat cells) and keratinocytes (skin cells). Keratinocytes may be preferred. It may be a stem cell that is usable for repair of tissue, such as non-skeletal and skeletal tissues. The stem cells may be allogenic or autologous bone marrow MSCs (BMSCs). The stem cells may be those that have been generated by parthenogenesis. The "fluorescent conjugated polymer nanodot" may be a "fluorescent polymer (nano)dot" comprising one or more fluorescent conjugated polymers. It may additionally comprise one or more amphiphilic moieties.

The fluorescent conjugated polymer may be a polymer comprising a π-electron delocalized backbone. The conjugated polymer may be partially or fully conjugated. The fluorescent conjugated polymer may be a chromophoric polymer which allows selective light absorption and results in characteristic colouration.

The fluorescent conjugated polymer may be a homopolymer, a blend of polymers or a copolymer. The fluorescent conjugated polymer may comprise repeating units of optionally substituted cyclic group linked to O-containing heteroaryl group.

The optionally substituted cyclic group may be monocylic or polycyclic. The optionally substituted cyclic group may be aromatic and may be selected from the group consisting of fluorene groups, phenylene groups, thiophene groups, carbazole groups, and boron- dipyrromethene groups. The O-containing heteroaryl group may be monocylic or polycylic and may be selected from the group consisting of diazole, benzodiazole, oxazole, benzooxazole , pyran, furan, and benzofuran. A benzofurazan (2,1,3-benzoxadiazole) moiety may be especially mentioned.

The fluorescent conjugated polymer may therefore be selected from the group consisting of fluorene polymers, phenylene vinylene polymers, phenylene polymers, phenylene ethynylene polymers, thiophen polymers, carbazole fluorene polymers, boron-dipyrromethene-based polymers, and polymer blends and copolymers thereof .

The fluorescent conjugated polymer may be poly (9,9-dihexylfluorene-alt-2,l,3- benzoxadiazole) (PFBD) represented by the following formula:

[CHEM. 1]

It is a known fluorescent conjugated polymer (Y. Li , J. Liu , B. Liu , N. Tomczak, Nanoscale, 2012,4, 5694-5702).

Advantageously, when PFBD is selected as the conjugated polymer, the absorption maximum of PFBD may advantageously perfectly match the 488 nm laser equipped on a confocal laser scanning microscope. Advantageously, the extinction coefficient of the presently disclosed

PFBD-containing nanodots at 488 nm may be 1.44 x 10 8 M -1 cm -1. According to one embodiment the fluorescent conjugated polymer nanodot comprises a conjugated polymer capped with an amphiphilic co-polymer. The amphiphilic co-polymer may be selected from the group consisting of derivatives of polyethylene glycol (PEG), polypropylene glycol, polybutylene glycol, polycarboxymethylene, polycarboxyethylene (or polyacrylic acid (PAA) ) , polycarboxypropylene and polycarboxybutylene. The hydrophilic end of the amphiphilic co-polymers may comprise hydrophilic polymers. The hydrophilic polymers may be selected from the group consisting of polyoxyalkylene, polyalkylene glycol and polycarboxyalkylene .

The amphiphilic co-polymer may preferably be a poly(ethylene glycol) (PEG) derivative with a molecular weight of about 500 to 50,000. The molecular weight of PEG may preferably be in the range of about 1500 to about 5000, or about 1800 to about 4800, or about 1800 to about 4600, or about 1800 to about 4400, or about 1800 to about 4200, or about 1800 to about 4000, or about 1800 to about 3800, or about 1800 to about 3600, or about 1800 to about 3400, or about 1800 to about 3200, or about 1800 to about 3000, or about 1800 to about 2800, or about 1800 to about 2600, or about 1800 to about 2400, or about 1800 to about 2200, or about 1800 to about 2000, or about 2000 to about 5000, or about 2200 to about 5000, or about 2400 to about 5000, or about 2600 to about 5000, or about 2800 to about 5000, or about 2800 to about 5000, or about 3000 to about 5000, or about 3200 to about 5000, or about 3400 to about 5000, or about 3600 to about 5000, or about 3800 to about 5000, or about 4000 to about 5000, or about 4200 to about 5000, or about 4400 to about 5000, or about 4600 to about 5000, or about 4800 to about 5000, or about 1800, about 2000, about 2200, about 2400, about 600, about 2800, about 3000, about 3200, about 3400, about 3600, about 3800, about 4000, about 4200, about 4400, about 4600, about 4 00, or about 5000.

If a PAA derivative is used , the molecular weight of PAA may be in the range of about 2000 to about 5000, or about 2000 to about 4800, or about 2000 to about 4600, or about 2000 to about 4400, or about 2000 to about 4200, or about 2000 to about 4000, or about 2000 to about 3800, or about 2000 to about 3600, or about 2000 to about 3400, or about 2000 to about 3200, or about 2000 to about 3000, or about 2000 to about 2800, or about 2000 to about 2600, or about 2000 to about 2400, or about 2000 to about 2200, or about 2000 to about 5000, or about 2200 to about 5000, or about 2400 to about 5000, or about 2600 to about 5000, or about 2800 to about 5000, or about 2800 to about 5000, or about 3000 to about 5000, or about 3200 to about 5000, or about 3400 to about 5000, or about 3600 to about 5000, or about 3800 to about 5000, or about 4000 to about 5000, or about 4200 to about 5000, or about 4400 to about 5000, or about 4600 to about 5000, or about 4800 to about 5000, or about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about 3200, about 3400, about 3600, about 3800, about 4000, about 4200, about 4400, about 4600, about 4800, or about 5000.

The amphiphilic co-polymer may be a phospholipid PEG conjugate. The lipid moiety in the conjugate may be a l,2-distearoyl-sn-glycero-3-phosphoethanolamino (DSPE) moiety, a 1,2- Dimyristoyl-sn- glycero-3 -phosphoethanolamine (DMPE) moiety, a 1 ,2-Dilauroyl-sn-glycero- 3 -phosphoethanolamine (DLPE) moiety, or a l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE) moiety. The amphiphilic co-polymer may be selected from the group consisting of 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamino-N- [(polyethylene glycol)-2000]-(DSPE-PEG), 1 ,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine N-[(polyethylene glycol)-2000]-(DMPE-PEG) , l,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]-(DLPE-PEG), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]-(DPPE- PEG), l,2-distearoyl-sn-glycero-3-phosphoethanolamino-N- [amino (polyacrylic acid)]- (DSPE- PAA), l,2-distearoyl-sn-glycero-3-phosphoemanolamino-N-[amino(poly acrylic acid)]- (DMPE- PAA), l,2-distearoyl-sn-glycero-3-phosphoethanolamino-N- [amino (polyacrylic acid)]- (DLPE- PAA) and l,2-distearoyl-sn-glycero-3-phosphoethanolamino-N-[amino(polyacrylic acid)]- (DPPE- PAA) derivatives.

The amphiphilic co-polymer may be selected from the group consisting of: 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DSPE-PEG₂ooo)_> 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE- PEG2000-OCH₃ ), l,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG2000-NH2), l,2-disteardyl-sn-glycero-3-phosphoethanolamine- N- [carboxy (polyethylene glycol)-2000] (DSPE-PEG2000-COOH) , l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2ooo-maleimide), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2ooo-biotin), l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DMPE-PEG2000), l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (DMPE-PEG2000-OCH₃) , l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DMPE-PEG2000-NH2) , 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene glycol)-2000] (DMPE-PEG2000-COOH), l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DMPE-PEG2ooo-maleimide), l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DMPE-PEG2ooo-Biotin), 1,2- Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[ (polyethylene glycol)-2000] (DLPE-PEG2000) , 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DLPE-PEG2000-OCH₃), l,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DLPE-PEG2000-NH2), l,2-Dilauroyl-sn-glycero-3- phosphoethanolamine-N-[carboxy (polyethylene glycol) -2000] (DLPE-PEG2000-COOH), 1,2- Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) -2000] (DLPE-PEG2ooo-maleimide) , 1 ,2-Dilauroyl-sn-glycero-3 -phosphoethanolamine-N-

[biotinyl(polyethylene glycol)-2000] (DLPE-PEG2ooo-Biotin), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[ (polyethylene glycol) -2000] (DPPE-PEG2000), 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000] (DPPE-PEG2000- OCH₃), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N- [amino (polyethylene glycol)- 2000] (DPPE-PEG2000-NH2), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene glycol)-2000] (DPPE-PEG2000-COOH), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DPPE-PEG2ooo-maleimide), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[biotinyl (polyethylene glycol)-2000] (DPPE-PEG2ooo-Biotin), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ (polyacrylic acid)] (DSPE-PAA), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyacrylic acid)] (DSPE-PAA-OCH₃), l,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyacrylic acid)] (DSPE-PAA -NH₂), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carboxy (polyacrylic acid)] (DSPE-PAA-COOH), 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[maleimide (polyacrylic acid)] (DSPE-PAA-maleimide), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl (polyacrylic acid)] (DSPE-PAA- biotin), l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[ (polyacrylic acid) ] (DMPE- PAA); l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyacrylic acid)] (DM PE- PAA-OCH₃), l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[amino (polyacrylic acid)] (DMPE-PAA- NH₂) , l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[carboxy (polyacrylic acid)] (DMPE- PAA-COOH), l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide (polyacrylic acid)] (DMPE-PAA-maleimide), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl (polyacrylic acid)] (DMPE- PAA- Biotin), 1, 2-Dilauroyl-sn-glycero-3-phosphoethanolamine- N-[ ( polyacrylic acid)] (DLPE- PAA), l,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyacrylic acid)] (DLPE-PAA-OCH₃), 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyacrylic acid)] (DLPE-PAA-NH₂), 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyacrylic acid) ] (DLPE-PAA,-COOH), l,2-Dilauroyl-sn-glycero-3-phosphoethanolamine- N-[maleimide (polyacrylic acid)] (DLPE-PAA-maleimide), l,2-Dilauroyl-sn-glycero-3- phosphoethanolamine-N-[biotinyl (polyacrylic acid)] (DLPE-PAA-Biotin), 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy (polyacrylic acid)] (DPPE- PAA-OCH₃), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyacrylic acid)] (DPPE-PAA - NH₂), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[carboxy (polyacrylic acid)] (DPPE-PAA-COOH), l,2-dipalmitoyl-sn.-glycero-3-phosphoethanolamine-N-[maleimide (polyacrylic acid)] (DPPE-PAA-maleimide) and l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N- [biotinyl (polyacrylic acid)] (DPPE- PAA-Biotin). More than one type of amphophilic co-polymer may be present in the inventive nanodots, for example, the flourescent may comprise multiple amphiphilic molecules selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphoethanolamino-N-[(polyethylene glycol)-2000] (DSPE-PEG2000), l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[ (polyethylene glycol)- 2000] (DMPE-PEG2000), 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DLPE-PEG2000), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [(polyethylene glycol)-2000] (DPPE-PEG2000), l,2-distearoyl-sn-glycero-3- phosphoethanolamino-N- [amino (polyacrylic acid) ] (DSPE-PAA), l,2-distearoyl-sn-glycero-3- phosphoethanolamino-N- [amino (polyacrylic acid)] (DMPE-PAA), l,2-distearoyl-sn-glycero-3- phosphoethanolamino-N- [amino (polyacrylic acid)] (DLPE-PAA), 1, 2-distearoyl-sn-glycero-3- phosphoethanolamino-N- [amino (polyacrylic acid)] (DPPE-PAA) derivatives and combinations thereof. An embodiment of the invention where the amphiphilic co-polymer is a mixture of at least one l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol with maleimide functionality and at least one l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol each having a molecular weight between 750 and 10,000 can be especially mentioned. As components of this mixture DSPE-PEG₂ooo and DSPE- PEG₂oo-maleimide may be especially mentioned.

The nanodots may have a have a core-shell-structure comprising a hydrophobic core and hydrophilic segments forming a protective shell layer. The hydrophobic core of said nanodot may comprise the conjugated polymer and the hydrophilic shell may comprise the hydrophilic segment of the amphiphilic co-polymer. Each amphiphilic co-polymer may comprise a hydrophobic (lipophilic end) end embedded in the core and a hydrophilic end that forms a hydrophilic shell surrounding the core.

In one embodiment, the incorporation of DSPE-PEG and/or DSPE-PEG derivatives to PFBD can form a core-shell structure where hydrophilic PEG segments provide the protective shell layer and hydrophobic PFBD and hydrophobic DSPE form the hydrophobic core .

Advantageously, the lipid moiety may be biocompatible, thus making the disclosed flourescent CP nanodots safer for use in the stem cells.

Advantageously, the surface polymer segments may provide excellent colloidal stability in the disclosed nanodots which is critical in the stem cell tracking applications. In another embodiment, a biological molecule may be conjugated to the nanodot. The conjugation is preferably on the surface. The biological molecule may be selected from the group consisting of amino acids, peptides, polypeptides, nucleic acids, carbohydrates, lipids, fatty acids, antibodies, aptamers and proteins. The biological molecule may be conjugated to the surface of the nanodot via one or more abovementioned functional groups. The biological molecule may be cell penetrating peptide which facilitates cellular uptake of various molecular cargo. The cell penetrating peptide may be derived from HIV-1 transactivator of transcription (Tat) protein ('Tat Peptide)". The cell penetrating peptide may be Tat (RKKRRQRRRC) peptide (see S. Piantavigna , G. A. McCubbin , S. Boehnke , B. Graham , L. Spiccia, L. L. Martin , BBA Biomembr. 2011 , 1808 , 1811). The way in which the biological molecule may be attached to the surface of the nanodots is dependent on the type of functional group located on said surface. For example, attachments of peptides to fluorescent polymer nanodot-maleimide may be through known thiol-maleimide click reaction. Advantageously, the properties of biomolecule-conjugated functionalized nanodots in the stem cell tracking method may not change upon bioconjugation. The conjugation with the biomolecule may be performed in an aqueous solution of the synthesized nanodots in the presence of co-solvents, such as DMSO or DMF. The weight ratio of conjugated polymer to amphiphilic co-polymer may be in the range of about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 65%, about 25% to about 30%, about 30% to about 75%, about 35% to about 75%, about 40% to about 75%, about 45% to about 75%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, or about 70% to about 75%. The weight ratio of conjugated polymer to amphiphilic polymer may be about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%. The weight ratio of conjugated polymer to amphiphilic molecule may be about 50%.

The flourescent CP nanodots can be made according one embodiment of the invention by preparing a mixture of fluorescent conjugated polymer and amphiphilic copolymer in an aprotic solvent and adding said mixture to a protic solvent, to form the polymer nanodot. The aprotic solvent may be selected from the group consisting of tetrahydrofuran, ether, dichloromethane, acetone, acetonitrile, DMF, and the mixtures thereof. The aprotic solvent may be tetrahydrofuran. The protic solvent may be selected from lower alcohols, water, and mixtures thereof. The protic solvent may be deionized (DI) water.

The mixture of conjugated polymers and amphiphilic co-polymer in aprotic solvent may be quickly added to a protic solvent to achieve a preferable nandot structure of the desired size. The method further may comprise the step of controlling the size of the Nanodots produced. The step may comprise modifying the initial concentration of conjugated polymer in aprotic solvent.

The initial concentration of conjugated polymer in aprotic solvent may be in the range of about 0.05 mg/mL to about 1.50 mg/mL, about 0.10 mg/mL to about 1.50 mg/mL, about 0.20 mg/mL to about 1.50 mg/mL, about 0.30 mg/mL to about 1.50 mg/mL, about 0.40 mg/mL to about 1.50 mg/mL, about 0.50 mg/mL to about 1.50 mg/mL, about 0.60 mg/mL to about 1.50 mg/mL, about 0.70 mg/mL to about 1.50 mg/mL, about 0.80 mg/mL to about 1.50 mg/mL, about 0.90 mg/mL to about 1.50 mg/mL, about 1.00 mg/mL to about 1.50 mg/mL, about 1.10 mg/mL to about 1.50 mg/mL, about 1.20 mg/mL to about 1.50 mg/mL, about 1.30 -mg/mL to about 1.50 mg/mL, about 1.40 mg/mL to about 1.50 mg/mL, about 0.05 mg/mL to about 1.40 mg/mL, about 0.05 mg/mL to about 1.30 mg/mL, about 0.05 mg/mL to about 1.20 mg/mL, about 0.05 mg/mL to about 1.10 mg/mL, about 0.05 mg/mL to about 1.00 mg/mL, about 0.05 mg/mL to about 0.90 - mg/mL, about 0.05 mg/mL to about 0.80 mg/mL, about 0.05 mg/mL to about 0.70 mg/mL, about 0.05 mg/mL to about 0.60 mg/mL, about 0.05 mg/mL to about 0.50 mg/mL, about 0.0£T mg/mL to about 0.40 mg/mL, about 0.05 mg/mL to about 0.30 mg/mL, about 0.05 mg/mL to about 0.20 mg/mL, or about 0.05 mg/mL to about 0.10 mg/mL. The initial concentration may be about 0.05 mg/mL, about 0.10 mg/mL, about 0.20 mg/mL, about 0.30 mg/mL, about 0.40 mg/mL, about 0.50 mg/mL, about 0.60 mg/mL, about 0.70 mg/mL, about 0.80 mg/mL, about 0.90 mg/mL, about 1.00 mg/mL, about 1.10 mg/mL, about 1.20 mg/mL, about 1.30 mg/mL, about 1.40 mg/mL, or about 1.50 mg/mL. In one embodiment the amphiphilic co-polymer is a mixture of at least one 1,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol with maleimide functionality and at least one l ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol each having a molecular weight between 750 and 10,000 and their concentration is in aprotic solvent may be each in the range of about 0.05 mg/mL to about 1.50 mg/mL, about 0.10 mg/mL to about 1.50 mg/mL, about 0.20 mg/mL to about 1.50 mg/mL, about 0.30 mg/mL to about 1.50 mg/mL, about 0.40 mg/mL to about 1.50 mg/mL, about 0.50 mg/mL to about 1.50 mg/mL, about 0.60 mg/mL to about 1.50 mg/mL, about 0.70 mg/mL to about 1.50 mg/mL, about 0.80 mg/mL to about 1.50 mg/mL, about 0.90 mg/mL to about 1.50 mg/mL, about 1.00 mg/mL to about 1.50 mg/mL, about 1.10 mg/mL to about 1.50 mg/mL, about 1.20 mg/mL to about 1.50 mg/mL, about 1.30 -mg/mL to about 1.50 mg/mL, about 1.40 mg/mL to about 1.50 mg/mL, about 0.05 mg/mL to about 1.40 mg/mL, about 0.05 mg/mL to about 1.30 mg/mL, about 0.05 mg/mL to about 1.20 mg/mL, about 0.05 mg/mL to about 1.10 mg/mL, about 0.05 mg/mL to about 1.00 mg/mL, about 0.05 mg/mL to about 0.90 - mg/mL, about 0.05 mg/mL to about 0.80 mg/mL, about 0.05 mg/mL to about 0.70 mg/mL, about 0.05 mg/mL to about 0.60 mg/mL, about 0.05 mg/mL to about 0.50 mg/mL, about 0.0£T mg/mL to about 0.40 mg/mL, about 0.05 mg/mL to about 0.30 mg/mL, about 0.05 mg/mL to about 0.20 mg/mL, or about 0.05 mg/mL to about 0.10 mg/mL. The initial concentration may be about 0.05 mg/mL, about 0.10 mg/mL, about 0.20 mg/mL, about 0.30 mg/mL, about 0.40 mg/mL, about 0.50 mg/mL, about 0.60 mg/mL, about 0.70 mg/mL, about 0.80 mg/mL, about 0.90 mg/mL, about 1.00 mg/mL, about 1.10 mg/mL, about 1.20 mg/mL, about 1.30 mg/mL, about 1.40 mg/mL, or about 1.50 mg/mL.

The mixture may be one of DSPE-PEG₂ooo and DSPE-PEG₂ooo maleimide. The amphiphilic copolymers in the mixture may be used in a molar ratio of 3: 1 to 1 :3, more preferably in substantially equimolar amounts. The amphiphilic copolymers may be used in excess or at lower amount than the conjugated polymer. In one embodiment they are used in substantially equimolar amount (CP : DSPE-PEG₂₀oo : DSPE-PEG₂₀oo maleimide = 1 : 1 : 1). The CP may be PFBD.

In one embodiment the method according to the invention comprises the following steps: (a) incubating the stem cells with the conjugated polymer nanodots or its encapsulated and\or functionalized derivatives; and (b) measuring the fluorescence at different times after incubation.

Step (a) involves typical cell incubation methods in a solution that may be buffered (e.g. with PBS buffer). Incubation times may range from about 5 to 24 hours, preferably about 1 to 7 hours, most preferably about 4 hours. The temperature may be about 36 to 38 °C. The concentration of the nanodots may be of about 0.1 to 50 nM, preferably about 1 to 10 nM, more preferably about 3 to 6 nM, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm. The incubation step may be followed by typical washing steps with buffer, detaching with an enzyme (e.g. trypsin) and subculturing of the labelled cells.

In the case of using PFBD he fluorescence profiles of cells can be recorded using flow cytometry by counting 10.000 events ( λ_6Χ= 488 nm, 575/25 nm bandpass filter). Advantageously, commercially available microscope, filter and camera systems can be used to visualize the luminescence of the labelled stem cells.

One embodiment of the invention may be a method as described above wherein the fluorescence is measured at different times over long periods after the tracking of about 1 to 28 days. Advantageously, the employed labelled stem cells can be well tracked over such long periods without any significant loss of intensities. A high labelling efficiency is retained for inventive methods that track the stem cells over periods of more than about 5, 10, 15, 20, 25, 28 or 60 days.

One embodiment of the invention may be a method comprising the steps of transplanting the labelled cells in an animal and tracking the transplanted stem cells development by repeated fluorescence measurements over time.

Advantageously, the employed labelled stem cells may be well tracked over extended times without influencing the stem cells development. The fluorescent conjugated polymer nanodots do not show toxicity to the cells or other negative influence. Therefore the methods according to the invention can be used as a research tool to evaluate the fate and development of stem cells over longer times which has not been possible so far.

One embodiment of the invention is a method for investigating the regeneration of skin wherein stem cells have been transplanted. This method may be used for observation over periods exceeding about 10 days, or most preferably exceeding about 15, 20, 25 or 60 days. Advantageously, the stem cells can develop well into tissue and can be researched in this development. This may give researchers very important knowledge about possible improvements in skin regeneration by using the method according to the invention in their research.

Another aspect of the invention is therefore a kit for stem cell tracking comprising a container with at least one fluorescent conjugated polymer nanodot (in pure form or preferably as its derivatives and conjugated form) together with other excipients for applying the nanodots to the cells or instructions on the use of the nanodots. Typical excipient will be physiological buffers (such as PBS) or dionized water. The kit will allow easy use of the new labelling molecules. Preferably a fluorescent conjugated polymer nanodot is selected from a nanodot comprising a fluorescent conjugated polymer capped with an amphiphilic co-polymer and conjugated to a Tat peptide. Preferably the concentration in a solution of the nanodot will be 1 to 10 nm.

A kit according to the invention wherein the amphiphilic co-polymer is a mixture of at least one l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol with maleimide functionality and at least one l,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE) conjugated polyethylene glycol each having a molecular weight between 750 and 10,000 may be especially mentioned Yet another aspect of the invention is a stem cell labelled with at least one fluorescent conjugated polyemer nanodot. The stem cell and the fluorescent conjugated polyemer nanodot are those described above in relation with the inventive method. Preferably a fluorescent conjugated polymer nanodot is selected from a nanodot comprising a fluorescent conjugated polymer capped with an amphiphilic co-polymer and conjugated to a Tat peptide.

Advantageously those stem cells can be visualized after transplantation and their behaviour investigated by researchers. The labelleing nanodot may have no disturbing influence on the stem cell development and proliferation and retain high effectiveness for visualisation over extended times.

Brief Description of Drawings

The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

Fig.l

[Fig. 1] shows flow cytometry histograms of MSCs after incubation with 4 nM (a) Tat- PFBD nanodots and (b) Qtracker® 585 at 37 °C for 4 hours and then subcultured for designated time intervals. The untreated MSCs were used as the control. In Fig. 1(a), the legend is as follows: control (100), Day 0 (102), Day 2.5 (104), Day 5 (106), Day 10 (108), Day 15 (110) and Day 25 (112). In Fig. 1(b), the legend is as follows: control (120), Day 0 (122), Day 2.5 (124), Day 5 (126) and Day 10 (128).

Fig.2 [Fig. 2] shows (a) the proliferation profiles of MSCs incubated with 4 nM Tat-PFBD nanodots for 4 h and subcultured for 3, 6 and 9 days in fresh medium using nanodot-free MSCs as control; (b) the metabolic viabilities of MSCs after incubation with 4, 6 and 8 nM Tat-PFBD nanodots for 48 and 72 hours, respectively; (c) the migration of Tat-PFBD nanodot-labeled MSCs on coverslips in the wound healing assay at 0, 12 and 24 hours after creating wounds. Nuclei (blue) and actin filaments (red) were visualized by DAPI and Alexa Fluor® 633 phalloidin staining. Scale bar = 300 μπι.

Fig. 3

[Fig. 3] shows (a) a real-time PCR results of KRT10 and FLG mRNA expression levels in Tat-PFBD nanodot-labelled (black) and unlabelled MSCs (white) that were cultured in epidermal induction medium after 15 days; (b) the mean traction stress of Tat-PFBD nanodot-labelled (black) and unlabelled MSCs (white) on day 0 and day 15 (n = 10) ; (c) representative phase contrast images superimposed with colour coded traction stress maps of Tat-PFBD nanodot-labelled and unlabelled MSCs on day 0 and day 15. Scale bar = 10 μπι; (d) secretome analyses of MSCs with and without Tat-PFBD nanodot labelling as well as dermal fibroblast.

Fig. 4

[Fig. 4] shows (a) representative in vivo fluorescence images of the wound sites on mouse transplanted with 1 x 10⁶ of GFP/luciferase double-expressing MSCs labelled with Tat- PFBD nanodots (left side wound) and that without nanodot labelling (right side wound) (n = 8 for each group); (b) time- dependent fluorescence intensity changes for the region of interest (ROI): the wound sites were identified by red and blue circles in (a) ; (c) corresponding in vivo luminescence images of the same mouse in (a) ; (d) Ttme- dependent fluorescence intensity changes for the ROI in (c); (e) percentages of epithelialized wound area in different groups from day 0 to 21. The digital photos show the wound sites of different groups at day 21. (The scale bar is 1 cm); (f) histological analysis of skin tissues from the wound site treated with Tat-PFBD nanodot-labelled MSCs, unlabelled MSCs, Matrigel and saline after 7 and 21 days.

Fig. 5 [Fig. 5] shows confocal images of the regenerated skin tissue sections from mice treated with (a) MSCs, (b) saline, and (c) Tat-PFBD nanodot-labelled MSCs, respectively (The tissue sections were immunostained with KRT10 and FLG antibodies and reveal by Alexa Fluor® 633. The generated epidermis was highlighted with dashed line. Scale bar is 100 μηι.); (d) the enlarged confocal imaging indicating the engraftment of Tat-PFBD nanodot- labelled MSCs in the regenerated skin; (e) 3D confocal images of the regenerated skin tissue collected from mouse treated with Tat-PFBD nanodot-labelled MSCs (The red fluorescence is from GFP antibody/ Alexa Fluor® 633 and yellow signal is from Tat-PFBD nanodots in the transplanted MSCs. The nuclei were stained with DAPI.)

Fig. 6 [Fig. 6] shows schematic syummary picture of a preferred embodiment of the inventive method.

Examples

Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention. Example 1: Preparation of the fluorescent CP nanodots

Materials: PFBD was synthesized as mentioned in the description. DSPE-PEG2000 and DSPE-PEG2000 -Mal(eimide) were purchased from Avanti Polar Lipids, Inc. DMEM (low glucose), DMEM/nutrient mixture F12 (DMEM/F12), FBS, penicillin-streptomycin solution, 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), Alexa Fluor 633 Phalloidin, and Qtracker 585 were all purchased from Life Technologies, Invitrogen, Singapore. Human bone marrow-derived MSCs were obtained from Lonza (Portsmouth, NH). Tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ascorbic acid, epidermal growth factor (EGF), 1, 25 -dihydroxy vitamin D 3 (VD 3), hydrocortisone, insulin, and 3,3, 5-triiodo-L -thyronine sodium (T3) were all purchased from Sigma- Aldrich (Singapore). The RNeasy Mini Kit and the GAPDH, KRT10 and FLG primers were all purchased from Qiagen, Singapore. Cell penetrating peptide, HIV- 1 Tat (50-57) with cysteine-modified terminus (RKKRRQRRRC), was customized by GenicBio, China.

Synthesis of Tat-PFBD Nanodots: A homogeneous THF solution containing PFBD, DSPE-PEG 2000 and DSPE-PEG 2000 -Mal(eimide) (1 mg each) was quickly injected into water, followed by continuous sonification using a probe sonicator at 12 W output (XL2000, Misonix Incorporated, NY). The suspension was then stirred vigorously at room temperature overnight to evaporate THF and yield PFBD nanodots in water (5 mL). The formed suspension was then filtered through a 0.2 urn syringe filter. The nanodots (2 mL) were further mixed with HIVl-Tat peptide (5 x 10 ²M ) in the presence of DMSO (1 x 10 ^~ M ). After overnight reaction at room temperature, the solution was dialyzed against MilliQ water for 2 days to eliminate the excess peptide. The PFBD nanodots were collected for further use and characterized.

In Vitro Cell Tracking: MSCs were cultured in six-well plates (Costar, IL, USA) to achieve 80% confluence. After medium removal and washing with lx PBS buffer, 4 x 10 ⁹M Tat-PFBD nanodots or Qtracker® 585 (as a reference) in DMEM medium was then added to the wells. After 4 hours incubation at 37 °C, the cells were washed twice with lx PBS buffer and detached by lx trypsin and resuspended in culture medium. Upon dilution, the cells were subcultured in six -well plates containing cell culture coverslips (diameter 15 mm) for 2.5, 5, 10, 15, and 25 days, respectively. After designated time intervals, the coverslips within the six-well plates were removed and fixed by 4% paraformaldehyde for 15 min. The coverslips were then sealed with mounting medium and the fluorescence images were studied by a confocal microscope (Olympus Fluoview FVIOOO). The rest of MSCs within six-well plates were washed twice with lx PBS buffer and detached by lx trypsin for resuspension in 4% paraformaldehyde. The fluorescence profiles of cells were then recorded using flow cytometry by counting 10 000 events ( λ_6Χ= 488 nm, 575/25 nm bandpass filter). In all flow cytometry tests, nanodot-free MSCs were used as the control. As shown in Fig. 1 a, the labelling rate of MSCs treated by Tat-PFBD nanodots was 99.9% at day 0 and the high labelling rate could be maintained up to 10 days (99.5%). After continuously culturing for 25 days, 41.7% of MSCs were still effectively labelled. On the contrary, the labelling efficiency of Qtracker® 585 -treated cells was significantly decreased from 99.6% (day 0) to 50.0% at day 5 and only 14.1% of the cells were detectable after 10 days (Fig. lb). These results demonstrated the superior cell tracking ability of Tat-PFBD nanodots over Qtracker® 585 in in vitro studies, due to the high labelling efficiency and stable fluorescence in biological environment.

Example 2: Cytotoxicity of Tat-PFBD Nanodots

Cytotoxicity of Tat-PFBD Nanodots: The metabolic activity of MSCs was evaluated by MTT assays to study the cytotoxicity of Tat-PFBD nanodots. MSCs were seeded in 96- well plates (Costar, IL, USA) at 2 x 10⁴ cells mL ^l . After 24 hour incubation, the medium was replaced by the Tat-PFBD nanodot suspension at concentrations of 4, 6, and 8 x 10^~9M , and the cells were then incubated for 48 and 72 hours, respectively. After the designated time intervals, the wells were washed twice with 1 * PBS buffer and 100 of freshly prepared MTT (0.5 mg mL ^l ) solution in culture medium was added into each well. The MTT medium solution was carefully removed after 3 hours incubation in the incubator at 37 °C. DMSO (100 μί) was then added into each well and the plate was gently shaken to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored.

In Vitro Proliferation and Migration Study: MSCs were cultured in T-25 flasks (Costar, IL, USA) to achieve 80% confluence. After medium removal and washing with lx PBS buffer, 4 x 10^~9 M Tat-PFBD nanodots in DMEM medium was then added to the flask. After 4 hours incubation at 37 °C, the cells were washed twice with lx PBS buffer and detached by lx trypsin and resuspended in culture medium. Upon dilution, the cells were seeded in 24-well plates at a density of 5 x 10³ cells/well. MSCs without nanodots treatment were used as control. The cells were cultured for 3, 6 and 9 days. After designated time intervals, MTT assays were carried out to evaluate the metabolic activity of MSCs, compared to the untreated MSCs. MSC migration was evaluated using the monolayer scratch wound assay. The Tat-PFBD nanodot-labeled and unlabelled MSCs were seeded onto a round coverslips (15 mm diameter) located in a 24-well plate, and cultured until confluence. Next, the monolayer of MSCs was wounded with a 10 μL· pipette tip, followed by washing with lx PBS to eliminate the detached cells. Upon incubation for 12 and 24 hours in fresh medium, the cells were fixed with 4% paraformaldehyde in PBS for 15 min and stained by DAPI/Alexa Fluor 633 phalloidin for fluorescence visualization of nuclei/actin filament. Images of the wounds were acquired by a confocal microscope (Olympus Fluoview FVlOOO). Impact of Tat-PFBD nanodots on MSC behaviours: The ideal fluorescent cell trackers are expected to have minimal perturbation to the functions of transplanted stem cells that are generally sensitive and fragile to exterior stimuli. To address this issue, the behaviours (proliferation, migration, differentiation and paracrine signalling) of MSCs treated with Tat- PFBD nanodots were investigated first, using nanodot-free MSCs as control. The in vitro proliferation of MSCs after labelling with 4 nM Tat-FPBD was firstly evaluated by methylthiazolyl-diphenyl-tetrazolium bromide (MTT) assay. The cell metabolic activity results revealed that Tat-PFBD nanodot -labelled MSCs had similar cell number with the nanodot-free MSCs after further incubation in fresh medium for 3, 6 and 9 days, confirming the low cytotoxicity of Tat-PFBD nanodots to MSCs. Furthermore, the motility of MSCs treated by Tat- PFBD nanodots was also investigated through an in vitro wound healing model, indicating that the MSCs with or without nanodot labelling showed similar wound healing progress, suggesting that Tat-PFBD nanodots had negligible effect on the cell migration ability (see Fig. 2). Cell Traction Force Microscopy:

The polyacrylamide gel films with embedded fluorescent microspheres were prepared according to literature and used for CTF microscopy study. Briefly, a mixture of 2.5% acrylamide (Bio- Rad), 0.5% bis-acrylamide (Bio-Rad), tetramethylethylenediamine (TEMED, Bio-Rad), ammonium persulfate (APS, Bio-Rad), and fluorescent polystyrene microbeads (diameter 0.1 μιη, Invitrogen) were added onto glass coverslips. The 25 μΐ_^ droplet was covered by another coverslip, which was removed after polymerization. The gel substrate was «70 μιη thick with a Young's elastic modulus of 10 kPa. Before cell seeding, the formed PAA gelfi lm was coated with 50 μg h ¹ of human plasma fi bronectin (Sigma- Aldrich) overnight. To map the traction stress cell exerted on the substrate, the deformation field of the gel substrate was fi rst determined by tracking fl uorescent microbeads embedded right below the surface of the gel substrate. The displacement fi eld of the microbeads was obtained by comparing images of the fl uorescent microbeads before and after cell trypsinization. The deformation fi eld of the gel substrate was then translated into traction stress fi eld on the basis of Green's function. Briefly, the traction force at discrete point f located at the position (x_;, y_;) was calculated based on the following formulation: u_t ^*(x, y) =∑ ₌₁ G (x— x_t, y— ^A where G denotes the Greens' tensor and u denotes the experimental displacements of fluorescent beads at position (x_;, y_;). The overall force of the cell (F) is an integral of the traction filed magnitude over the area, F =

// where T( x,y) = [T_x( x,y)+ T_y{ x,y)] is the continuous field of

traction vectors defined at any spatial position (x, y) in the cell. The average level of traction stress was calculated as overall traction forces divided by the area. The cell traction stress map was then integrated with the differential interference contrast (DIC) image of cells on gel substrate, and the corresponding traction stress was mapped in pseudocolor, which indicated regions of traction stresses (dark blue to light pink according to stresses from low to high).

In Vitro Differentiation Study: MSCs were induced to epidermal lineages to assess the effect of Tat-PFBD nanodots on stem cell differentiation. 4 x 10 ⁹ M Tat-PFBD nanodots-labeled and unlabeled MSCs were seeded onto six-well plates at 1 x 10⁴ cells mL^-1. After culturing overnight, the medium was replaced with epidermal induction medium comprising DMEM and Ham's F12 medium (3: 1) including 10% FBS, 100 IU ml/¹ penicillin, and 100 μg m^"1 streptomycin, supplemented with 0.4 μg mL^of hydrocortisone, 5 μg mL ¹ of insulin, 1 x 10 ⁹ M of T3, 10 ng mL ¹ of EGF, 1 x 10^~6 M of VD 3 , and 50 μg mL ¹ of L -ascorbic acid. All samples were incubated under standard culture conditions of 37 °C in a sterile humidified incubator with 5% C0₂ and the culture medium was changed every 4 days. After 15 days, quantitative evaluation of the targeted KRT10 and FLG mRNA was assessed by RT-PCR. RNA from each sample was extracted with the RNeasy Mini Kit (Qiagen, Singapore), according to the manufacturer's instruction. 500 ng of extracted RNA was used to synthesize cDNA by the Superscript III kit (Invitrogen). Endogenous mRNA levels were measured by real-time PCR analysis based on SYBR Green detection with an ABI real-time PCR machine. The KRT10 and FLG primer pairs used were QuantiTect primer assays (Qiagen). Samples were analyzed three times and normalized to GAPDH.

Secretome Analyses of MSCs with and without Tat-PFBD Nanodot Labeling: Stem cells were incubated with Tat-PFBD nanodots and continued to culture for 5 days and then cells were analyzed by realtime PCR. Total RNA was extracted with TranZol Reagent (TransGen Biotechnology, Beijing, China). The PCR was performed in triplicate with the FastStart Universal SYBR Green Master (ROX; Roche, Mannheim, Germany) and ran on the iCycler iQ52.0 Standard Edition Optical System (Bio-Rad, Hercules, CA, USA). Primers used in PCR include VEGF-A, SDF-1, TGF-βΙ, bFGF, IL-6, MCP-1, and Ang-1. The results were analyzed against the housekeeping gene GAPDH.

Results: Considering the capabilities of self-renewal and pluripotent differentiation are key factors for MSCs in regenerative medicine, the differentiation of Tat-PFBD nanodot-labelled stem cells was quantitatively analyzed by reverse transcription polymerase chain reaction (RT- PCR). An early marker of epidermal differentiation, keratin 10 (KRT10), and an intermediate marker of epidermal differentiation, filaggrin (FLG), were chosen for analysis. Tat-PFBD nanodot-labelled MSCs and nanodot-free MSCs were separately cultured in an epidermal induction medium for 15 days, followed by extraction of total RNA with the RNeasy Mini Kit, using KRT10 and FLG primers to analyze the relative endogenous mRNA. The Tat-PFBD nanodot-treated MSCs and nanodot-free MSCs showed no significant difference in expression levels of KRT10 and FLG mRNA (Fig. 3a), confirming that the internalization of Tat-PFBD nanodots did not suppress MSC epidermal differentiation.

In addition, high-resolution traction force microscopy was used to determine the spatial distribution and dynamics of traction force in Tat-PFBD nanodot-labelled MSCs, which could provide insight into the impact of nanodot internalization on cell differentiation from a biophysical point of view. MSCs with or without incubation of 4 nM Tat-PFBD nanodots for 4 hours were separately seeded on fibronectin-coated polyacrylamide hydrogel impregnated with red fluorescent beads and cultured in epidermal induction medium. At day 0 and day 15, cell tractions were quantified through measuring the displacements of fluorescent beads before and after trypsinization of the attached MSCs. Statistical analysis of several individual cells (n = 10) suggested that Tat-PFBD nanodot-labelled and unlabelled MSC had no significant difference in average cell traction stress at the same time points (Fig. 3b). The average traction stress of nanodot-labelled MSCs after differentiation (day 15) was -2.9 times as high as that before differentiation (day 0). Spatiotemporal mapping of cell traction stress revealed that the concentrated cell traction stress of a single cell has apparently changed from two ends of the spindle shaped cell to surrounding of the rectangle -shaped differentiated cell for both Tat-PFBD nanodot-labelled and unlabelled MSCs after differentiation (Fig. 3c).

Paracrine signalling is another primary mechanism for the beneficial effects of MSCs on skin regeneration through reducing inflammation, promoting angiogenesis, and inducing cell migration and proliferation. Thus, the secretome behaviours of Tat-PFBD nanodot-labelled and unlabelled MSCs were investigated by real-time PCR with nanodot-free dermal fibroblasts as reference. As shown in Fig. 3d, the levels of paracrine cytokines, including interleukin 6 (IL-6), vascular endothelial growth factor A (VEGF-A), angiopoietin 1 (Ang-1), stromal cell-derived factor 1 (SDF-1), monocyte chemotactic protein 1 (MCP-1), basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-βΙ), did not show significant difference from cells with or without Tat-PFBD nanodot labelling, verifying that the nanodot could not obviously affect the secretome behaviour of MSCs. Special attention should be paid to the cytokines (VEGF-A, Ang-1, SDF-1 and bFGF) that involve in angiogenesis. The expression levels of the four cytokines secreted from MSCs were significantly higher than that secreted from dermal fibroblasts, indicating that the paracrine signalling of MSCs is important in promoting skin regeneration. As a result, Tat-PFBD nanodots are ideal for tracking MSCs without compromising the cell behaviours of proliferation, migration, differentiation and secretome, which will benefit the long-term in vivo cell tracking studies.

Example 3: Tracking and engraftment evaluation of MSCs in mice with full-thickness wounds

Cell Culture : Luciferase+/GFP+ adipose-derived mesenchymal stemcells were isolated from the abdominal and inguinal adipose tissue of 8-12 weeks old male FVB-luc-GFP transgenic mice, which were subsequently cultured and expanded in 10 cm² plate in complete growth medium containing a-minimum essential medium supplemented with 10% FBS and 100 U mL - 1 of penicillin-streptomycin at 37 °C in a humidified environment containing 5% C0₂ . The culture medium was changed twice a week. Wound Healing Model and MSCs Transplantation: All animal studies were performed in compliance with the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals and the project protocols were approved by the Animal Ethics Committee of Nankai University. To create the mouse wound healing ischemic model, eightweek-old female BALB/c nude mice obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) were randomly divided into four groups, and the excisional wound splinting model was generated. In brief, after anesthesia, two 6 mm full-thickness excisional skin wounds were created on each side of the midline. Wound received: 1 x 10⁶ Tat-PFBD nanodot-labeled MSCs (Group I; n = 8), MSCs only (Group II; n = 8), Matrigel only (Group III; n = 8), and saline (Group IV; n = 8). For Tat-PFBD nanodot-labeled MSCs and nanodot-free MSCs transplantation, 5 x 10 ⁵ MSCs in 50 μL· of Matrigel (BD, Biosciences) were injected intradermally around the wound at four injection sites and 5 x 10^s MSCs in 10 μL· of Matrigel were applied onto the wound bed. For control groups, equal volumes of Matrigel and saline were implanted into excisional wounds in nude mice. A silicone splint was placed so that the wound was centered within the splint. An immediate -bonding adhesive (Krazy Glue, Columbus,OH, http://www.krazyglue.com) was used to fi x the splint to the skin, followed by interrupted sutures to stabilize its position, and Tegaderm (3M, London, ON, Canada, http://www.3m.com) was placed over the wounds. The animals were housed individually.

In Vivo Cell Tracking: Fluorescence images were taken using the Maestro in vivo Imaging System (CRI Inc., Woburn, MA) after 0, 5, 7, 14, 21, and 28 days. The detection was set to capture images automatically at 10 nm increments from 560 to 900 nm with constant 1 s exposure. The obtained tagged image file format (TIFF) images were loaded into the vendor's software and analyzed. After fluorescence imaging, the mice were administrated with a solution of luciferase substrate (D-luciferin) and the bioluminescence intensity generated from oxidation of luciferin was recorded on a Xenogen IVIS Lumina II system for 5 min. The same mice were scanned for one month. Imaging signals were quantified in units of maximum photons per second per square centimeter per steridian. Bioluminescence imaging was performed by a researcher blinded to the study conditions.

Wound Closure Analysis: Digital photographs of wounds were taken at day 0, 3, 7, 14 and 28. Time to wound closure was defined as the time at which the wound bed was completely re- epithelialized and filled with new tissue. Wound area was measured by tracing the wound margin and calculated using an image analysis program (NIH Image). The researchers who measured the samples were blinded to group and treatment. The percentage of wound closure was calculated as follows: [(Area of original wound - Area of actual wound)/Area of original wound] x 100%. The inside edge of the splint exactly matched the edge of the wound, so that the splinted hole was used to represent the original wound size. Mice were sacrificed at 7, 14, and 28 days, at which times, skin samples including the wound and 4 mm of the surrounding skin were harvested using a 10 mm biopsy punch.

Histology and Immunohistochemistry: The skins from the wound site of mice were excised at 1, 2, and 3 weeks and were fixed in 4 % paraformaldehyde for 24 hours for histological staining. The tissue specimens were embedded in paraffin or optimal cutting temperature (OCT) compound, and cut into 6 μπι thick sections, which were subjected to H&E staining. Images were observed under an inverted microscope (Nikon Eclipse TE2000-U Kanagawa, Japan) and analyzed by Nikon NIS Elements software. The regenerated skins from the wound site were also excised at 5, 14 and 21 days for and immunofluorescence staining. The fixed and frozen sections were stained with mouse antihuman KRT10 antibody (Thermo Fisher Scientific) and mouse antihuman FLG antibody (Thermo Fisher Scientifi c), respectively. Alexa Fluor 633 goat antimouse IgG (Life Technologies) was used as the secondary antibody to reveal KRT10 and FLG expression. The regenerated skin tissues from day 21 were further stained with rat anti- CD31 antibody (BD biosciences, USA), rabbit anti-PCNA antibody (Abeam, USA), rabbit anti- VEGF antibody (Abeam, USA), rabbit anti-GFP antibody (Abeam, USA), rabbit antibasic FGF antibody (Abeam, USA), and rabbit antibasic SDF-1 antibody (Abeam, USA). Alexa Fluor 633 goat antirabbit IgG (Life Technologies) and Alexa Fluor 633 goat antirat IgG (Life Technologies) were used as the secondary antibodies. The nuclei were stained with DAPI containing mounting solution (DAPI Fluoromount G, Southern Biotech, England). The sections without incubation with primary antibodies were used as negative controls. Slides were observed under confocal laser scanning microscopy (Leica TSC SP8, Germany). Results: The capability of Tat-PFBD nanodots for in vivo tracking and identifying the fate of transplanted MSCs was investigated using a full-thickness wound mouse model. A full-thickness excision wound (1 cm²) was generated on the left dorsal skin of each 8-week mice, followed by transplantation of 10⁶ MSCs labelled by Tat-PFBD nanodots using Matrigel as the substrate. A contralateral wound site on the same mice was also created on the right dorsal skin and transplanted with 10⁶ nanodot-free MSCs. Lucif erase and green fluorescent protein (GFP) double -expressing MSCs isolated from male FVB-luc-GFP transgenic mice were used in this study, which allowed in vivo monitoring of Tat-PFBD nanodot-labelled MSCs through both bioluminescence and fluorescence imaging to double confirm the reliability of Tat-PFBD nanodots in long-term cell tracking. Upon transplantation, intense fluorescence from the left wound site could be clearly distinguished at 1 hour post-wound infliction (PWI) (day 0) due to the bright emission from Tat-PFBD nanodots collected with a 560-900 nm filter (Fig. 4a). Quantitative analyses of the fluorescent signals from left wound site revealed a continuous decrease in fluorescence intensity during the skin regeneration (Fig. 4b). Meanwhile, the strong bioluminescence could be observed at both wound sites. The bioluminescence decay from both sites showed a homologous trend with fluorescence decay from the left wound site during the 21 days (Fig. 4c). As luciferase labeling strategy is well-known for its accuracy in living cell tracking, this result indicated the effectiveness and reliability of Tat-PFBD nanodots in long- term in vivo tracking of transplanted MSCs. Moreover, the bioluminescence intensity from transplanted MSCs with Tat-PFBD nanodot labelling showed similar bioluminescence to that from dot-free MSCs at each time point (Fig. 4d), suggesting that the internalization of Tat- PFBD nanodots has minimized influence on the bioluminescence imaging. The results further proved the priority of such organic nanodots in stem cell tracking as compared to inorganic Qtracker®, which was reported to greatly diminish the MSC function upon labelling and failed to conclusively track cell location during in vivo bone regeneration study The effect of Tat-FPBD labelling on regenerative function of MSCs during wound closure was studied using the mice with one full-thickness excision wound on the dorsal skin, followed by various treatments in four groups (n = 8 in each group). The sample group of mice were transplanted with Tat-PFBD nanodot-labelled MSCs on Matrigel in the wound sites, while the other three control groups of mice were separately treated with nanodot-free MSCs on Matrigel, saline or Matrigel. The appearance of wound closure was recorded by digital camera and the wounds on mice transplanted with nanodot-labelled and unlabelled MSCs were both completely healed at day 21 with similar progress (Fig. 4e). Additionally, both of the two groups showed faster wound closure comparing to the ones treated with saline or Matrigel, indicating the importance of MSCs in promoting wound healing and the negligible impact of Tat-PFBD nanodots on stem cell properties in long-term in vivo tracking. Hematoxylin and eosin (H&E) staining results showed that the wounds in the MSC-treated groups were totally covered by the newly formed skin after 7 days, in contrast to the open wounds in the tissue sections collected from Matrigel and saline treated groups (Fig. 4f). The newly formed epidermis and dermis with hair follicles (bluish droplets) were found in the MSCs treated groups after 21 days, while the formation of epidermis was still undergoing without hair follicles and sebaceous glands in the Matrigel and saline treated groups.

We further investigated the application of Tat-PFBD nanodots in understanding the roles of MSCs during skin regeneration through studying the engraftment of transplanted nanodot- labelled MSCs in regenerated tissues. To evaluate the epidermal differentiation potential of transplanted MSCs and the progress of regeneration of epidermis, the skin tissue sections were collected from all groups after 21 days for immunofluorescence staining. The results demonstrated that MSCs could accelerate wound healing process (Figs. 5a-c). After 21 days, the newly formed epidermis with similar expression of KRT10 and FLG were found in the skin sections collected from mice transplanted with MSCs (Fig. 5a). However, the epidermis in skin sections collected from the saline treated mice was not completely formed with voids in the epidermis layer (Fig. 5b). More importantly, the labelled MSCs were found to distribute within the dermis (Fig. 5c) and the enlarged image revealed that the internalized Tat-PFBD nanodots facilitated single cell identification with a bright yellow colour after 21 days upon transplantation (Fig. 5d). Especially, no KRT10 or FLG expression was found in Tat-PFBD nanodot-labelled cells (Fig. 5c), indicating that the transplanted MSCs did not undergo epidermal differentiation during skin regeneration. The accuracy and reliability of Tat-PFBD nanodots during in vivo tracking of MCSs were then examined by taking advantages of the GFP expression in cells. Upon amplifying signal through GFP antibody/ Alexa Fluor® 633 staining, The apparent co-localization of signals from Tat-PFBD nanodots (yellow) and GFP antibody/ Alexa Fluor® 633 (red) in cytoplasm suggested the ability of Tat-PFBD nanodots for precisely tracking the transplanted cells in long-term regenerative treatments (Fig. 5e). Quantitative analysis on day 21 based on 10 confocal images from different fields of regenerated skin tissues for each mouse (n = 8) indicated that -86% of GFP -expressed MSCs were stained with bright Tat-PFBD nanodots.

Fig. 6 shows a schematic drawing of a preferred embodiment of the inventive process as used in the examples of the invention.

Industrial Applicability

The method for tracking stem cells may find applications in in stem cell research. The method allows for imaging applications of stem cell development. Stem cells can be made visible for extended periods of time. The visualization may be useful for the screening of new and improved models for skin regeneration. The method is therefore important for biomedical industries in the field of stem cell based treatments where it is an important research tool in regenerative stem cell medicine industries. The use of the inventive method for diagnostic purposes on human beings or animals may be excluded.

Corresponding kits for such tracking purposes comprising fluorescent conjugated polymer nanodots may be an alternative to existing commercial stem cell tracking kids.

Stem cells labelled with the comprising fluorescent conjugated polymer nanodots may be commercially produced.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

1. A method for tracking stem cells characterized in that the stems cells are labelled with at least one fluorescent conjugated polymer nanodot and the fluorescence of the labelled cells is detected.

2. A method according to claim 1 wherein the stem cell is a mesenchymal stem cell.

3. A method according to claim 1 or 2, wherein the fluorescent conjugated polymer nanodot comprises a conjugated polymer capped with an amphiphilic co-polymer.

4. A method according to claim 3, wherein the amphiphilic co-polymer is a poly(ethylene glycol) (PEG) derivative with a molecular weight of about 500 to 50,000.

5. A method according to any of claims 3 to 4, wherein the amphiphilic co-polymer is a phospholipid PEG conjugate.

6. A method according to any of claims 3 to 5, wherein the amphiphilic co-polymer is a mixture of at least one l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol with maleimide functionality and at least one 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol each having a molecular weight between 750 and 10,000.

7. A method according to any of claims 3 to 6, wherein the amphiphilic co-polymer is a mixture of DSPE-PEG₂ooo and DSPE-PEG₂ooo maleimide.

8. A method according to any of claims 1 to 7 wherein the nanodot is conjugated to a cell penetrating peptide.

9. A method according to claim 8 wherein the cell penetrating peptide is a Tat peptide.

10. A method according to any of claims 1 to 9 wherein the nanodot has a particle size of about 5 to 50 nm.

11. The method according to any of claims 1 to 10 wherein the conjugated polymer has a benzofurazan (2,1,3-benzoxadiazole) moiety.

12. The method according to claim 11 wherein the conjugated polymer is poly(9,9- dihexylfluorene-alt-2, 1 ,3 -benzoxadiazole) (PFBD) .

13. A method according to any of claims 1 to 12 which comprises the following steps: (a) incubating the stem cells with the conjugated polymer nanodots or its encapsulated and\or functionalized derivatives; and

(b) measuring the fluorescence at different times after incubation.

14. A method according to claim 13 wherein the polymer nanodots or its encapsulated and/or peptide functionalized derivatives are used in step (a) at a concentration of about 0.1 to 50 nM and the incubation time is about 0.5 to 24 hours.

15. A method according to claim 13 or 14 wherein the fluorescence is measured at different times over long periods after the tracking of about 1 to 28 days.

16. A method according to claim 1 or 2 comprising the steps:

(a) transplanting the labelled cells in an animal,

(b) tracking the transplanted stem cells development by repeated fluorescence measurements.

17. Use of the method of claim 16 for investigating the regeneration of skin wherein stem cells have been transplanted.

18. A kit for stem cell tracking comprising a container with at least one fluorescent conjugated polymer nanodot together with other excipients for applying the nanodots to the cells or instructions on the use of the nanodots.

19. A kit according to claim 18 wherein the fluorescent conjugated polymer nanodot is selected from a nanodot comprising a fluorescent conjugated polymer capped with an amphiphilic co-polymer and conjugated to a Tat peptide.

20. A kit according to claim 19 wherein the amphiphilic co-polymer is a mixture of at least one l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol with maleimide functionality and at least one 1,2-Distearoyl-sn-

Glycero-3-Phosphoethanolamine (DSPE) conjugated Polyethylene Glycol each having a molecular weight between 750 and 10,000.

21. A stem cell labelled with at least one fluorescent conjugated polymer nanodot.