WO2011145963A1

WO2011145963A1 - Non-leaching nanoparticle formulation for the intracellular delivery of hydrophobic drugs and its use to modulate cell activity and differentiation

Info

Publication number: WO2011145963A1
Application number: PCT/PT2011/000017
Authority: WO
Inventors: Lino Da Silva Ferreira; João José OLIVEIRA MALVA; Liliana INÁCIO BERNARDINO; Tiago Alexandre RAMOS TEIXEIRA DE SOUSA SANTOS; João REINA MAIA E SILVA
Original assignee: Universidade De Coimbra
Priority date: 2010-05-20
Filing date: 2011-05-20
Publication date: 2011-11-24

Abstract

The present invention relates to polyelectrolyte nanoparticles able to induce neurogenesis only after being internalized by subventricular zone stem cells. The differentiated cells express typical neuronal markers including neuronal nuclear protein (NeuN), NMDA receptor subunit type 1 (NRl), and respond to the depolarization of KC1. The nanoparticles offer an opportunity for the in vivo delivery of pro-neurogenic factors and the treatment of neurodegenerative diseases. This system is focused on the delivery of hydrophobic small molecules, such as retinoic acid and other retinoids, 3, 3 ', 5, 5 ' -tetraiodo L-thyronine (T4), 3, 3 ', 5 ' -Triiodo-L- thyronine (T3) and others, which have the particularity of acting upon nuclear receptors. The proposed formulation is able to increase dramatically the drugs solubility, promote cell internalization of the drug and enhance its actuation. Most importantly, the formulation is able to mediate a cellular effect, only after cell internalization and not due to prior drug release.

Description

DESCRIPTION

"NON-LEACHING NANOPARTICLE FORMULATION FOR THE INTRACELLULAR DELIVERY OF HYDROPHOBIC DRUGS AND ITS USE TO MODULATE CELL

ACTIVITY AND DIFFERENTIATION"

FIELD OF THE INVENTION

The present invention is related to nanoparticle conjugates and methods of their preparation, in particular for the intracellular delivery of hydrophobic drugs, including retinoid acid and retinoid drugs. These nanoparticles can be used to control the activity and differentiation of cells, including stem cells, through the intracellular delivery of the hydrophobic drugs.

BACKGROUND OF THE INVENTION

Nanoparticles are on the scale of approximately 1 to 1000 nm in diameter. Representative examples of nanoparticles includes, organic and inorganic nanoparticles, liposomes, polyplexes, quantum dots and carbon nanotubes (Ferreira et al., Journal of Cellular Biochemistry 2009, 108, 746) .

Nanoparticles with a diameter ranging from few nanometers (2-10 nm, e.g. quantum dots) up to 1 micron can be taken up by stem cells or their progenies [Ferreira et al . , Advanced Materials 2008, 20:2285-2291]. The surface chemistry of the nanoparticles is also an important factor to control cellular uptake [Green et al., Nano Letters 2008,8:3126-30]. In most cases the nanoparticle charge provides the driving force for the uptake (through electrostatic interaction with the negatively charged cell membrane) and defines the internalization process. The nanoparticle charge also contributes for the adsorption of specific proteins in the cell medium that might enhance or reduce their cellular uptake. The ability to deliver biomolecules via an intracellular route, including proteins, growth factors, and small chemicals presents an excellent tool to control the activity and differentiation of cells, including stem cells. Some of these biomolecules/chemicals have (i) poor solubility, (ii) can be quickly cleaved by cellular enzymes, (iii) and have side effects if administered systemically . Biodegradable and biocompatible nanoparticles able to target cells and release the payload in their cytoplasm with consequent activation of signaling cascades will be of great interest. One of the inventors recently reported a new approach for the delivery of vascular growth factors into human embryonic stem cells, by incorporating growth factor-release particles in human embryoid bodies (EBs) [Ferreira et al., Advanced Materials 2008, 20:2285- 2291]. However, growth factors are typically hydrophilic molecules and therefore soluble in water for concentrations that are biologically effective. A major challenge remains to deliver intracellularly hydrophobic molecules. The present invention describes a nanoparticle formulation, and methods for its preparation, to deliver intracellularly hydrophobic molecules.

The term "hydrophobic" as used herein is taken to mean a compound that tends to be non-polar and thus prefer neutral or non-polar solvents. The "hydrophobicity" degree of a compound can be measured in a mixture of two immiscible solvents at equilibrium. Normally one of the solvents is water while the second is a hydrophobic solvent such as octanol . The differential solubility of the compound between 2 solvents is a meaure of its hydrophobicity. Therefore, compounds with a negative log P are hydrophilic compounds while the ones with a positive log P are hydrophobic compounds. Examples of hydrophobic compounds are found in oils, fats, phospholipids, small chemicals, anti-cancer agents, peptides, and other molecules that one skilled in the art will recognize. Some of these compounds and corresponding log P are listed in the table below.

Table - Log P values of several hydrophobic compounds (http: //pharmacycode . com)

The administration of hydrophobic compounds presents a significant challenge because of their poor water solubility, and potentially undesired side effects. One example is retinoic acid (RA) which has the ability to cross the nucleus membrane and interact with members of the hormone receptor super-family, including the RA receptor (PAR), located in the cell nuclei⁴. These receptors modulate the expression of a wide range of genes that act in cell proliferation, differentiation and apoptosis. Several studies have demonstrated that RA is able to induce the differentiation of stem cells into neuronal cells^4-6. However, RA is rapidly metabolized by cells and has low solubility in aqueous solutions⁷. In addition, the use of this biomolecule in an in vivo setting for the differentiation of resident stem cells remains elusive. Nanoparticles can be an excellent platform to ensure protection and intracellular transport of RA. Several nanoparticle formulations have been reported for the controlled release of RA^8"10; however, no study has demonstrated their ability to cross cell membrane and deliver RA intracellularly . Furthermore, no study has demonstrated that RA delivered intracellularly by nanoparticles could indeed control or modulate cell activity, and particularly stem cells.

Errico et al. (International Journal of Molecular Sciences 2009, 10, 2336) has developed RA-loaded nanoparticles formed by VA 41, an amphiphilic polymeric material developed by the hemiesterification of alternating copolymers of maleic anhydride and alkyl vinyl ethers. They tested the bioactivity of retinoic acid-loaded nanoparticles in a neuroblastoma cell line. The authors have shown qualitatively that retinoic acid-loaded nanoparticles could reduce the growth of neuroblastoma cell lines. However the authors did not show that the nanoparticles were indeed internalized by the cells and the biological effect was mediated by the intracellular release of RA.

Cells may take up nanoparticles via endocytosis, macropinocytosis , or phagocytosis, but these processes confine the internalized compounds to closed vesicles (endosomes or phagosomes), where the pH is progressively lowered to 5.5- 6.5¹¹'¹². Polycations, such as polyethylenimine, that absorb protons in response to the acidification of endosomes (i.e. cationic polymers with a pK around or slightly below physiological pH) can disrupt these vesicles via the "proton sponge" effect that promote the swelling of the particles in situ¹³'¹⁴. Once the polymeric nanoparticles reach the cytosol, the bioactive molecule may be released by desorption, diffusion through the nanoparticle, or nanoparticle erosion. - -

Errico, C. et al, "A novel method for the preparation of retinoic acid-loaded nanoparticles", Int. J. Mol . Sci. 2009, 10, 2336-2347, describe a nanoparticles containing retinoic acid but do not refer their ability to cross cell membrane and did not show that the bioactivity was truly mediated by the internalization of the nanopartiocle containing retinoic acid. Furthermore the authors have not demonstrate the bioactivity effect in more plastic cells such as stem cells. Thunemann, A., "Immobilization of Retinoic Acid by

Cationic Polyelectrolytes" , Langmuir, 1997, 13 (23), pp. 6040- 6046 discloses complexes of retinoic acid with cationic polyelectolytes ; however, they did not show their biological effect against cells. For those skilled in the art this effect is difficult to predict and not obvious.

WO 2007/115134 describes retinoid formulations with lipid nanoparticles. US 6,645,525 discloses a microparticle composition for delivery of a biomolecule.

BRIEF DESCRIPION OF THE DRAWINGS Figure 1 relates to Physico-chemical properties of the nanoparticles. A) Chemical structures of retinoic acid (RA) , polyethylenimine (PEI) and dextran sulfate (DS) . B) Transmission electron microscopy (TEM) images of RA+-NPs. Scale bar: 200 nm. C) Mass loss on nanoparticles suspended in buffered solutions at pH 5.0, 6.0 and 7.4 for 7 days, at 37 °C. Nanoparticles with or without RA were used in these degradation studies. Results are shown as mean + SD, n=6. ** and *** denotes statistical significance P<0.01 and P<0.001, respectively.

Figure 2 shows the diameter and release profile of RA^' - - containing nanoparticles. Diameter variation (A) and release profile (B) of RA^~containing nanoparticles suspended in buffered solutions at pH 5.0, 6.0 and 7.4 over 21 days, at 37°C. Figure 3 is related with the cellular nanoparticle uptake. A) Confocal live imaging of SVZ cells after exposure for 5 h to FITC-labeled RA+-NPs (50 pg/mL) . Hoechst-33342 staining (blue) was used to visualize cell nuclei. Crop images show both transmission and FITC channel or FITC channel alone. B) Confocal microscopy photomicrographs of untreated or FITC-labeled RA+- NPs-treated (10 pg/mL) SVZ cells for 18 h, imunostained with anti-FITC antibody and Hoechst-33342. Scale bars are 10 pm.

Figure 4 is related to the cell viability after RA^+/~- NPs uptake. A) Percentage of TUNEL-positive cells in control cultures and in cultures exposed to RA+-NPs for 2 days. Data are expressed as mean ± SEM (n=4-8 coverslips) . B) Percentage of PI- positive cells in control cultures and in cultures exposed to RA+-NPS or RA—NPs for 2 days. Data are expressed as mean ± SEM (n=4-9 coverslips). ***P<0.001 using Dunnet ¹ s . Multiple Comparison Test. C) Representative confocal microscopy photomicrographs showing a typical morphology of Tuj 1-positive neurons (red) , GFAP-positive glia (green) and Hoechst-33342 staining (blue nuclei) in SVZ cells maintained for 7 days in the absence (control) or in the presence of RA+-NPs (1 pg/mL) . Scale bars are 10 pm.

Figure 5 shows how the RA⁺-NPs have a proneurogenic effect. A) Representative fluorescence photomicrographs of NeuN immunostaining (red) in control cultures and cultures exposed to RA+-NPs (0.1 pg/mL) for 7 days. Hoechst 33342 was used for nuclear staining (blue) . B) Percentage of NeuN-immunostained neurons in SVZ cultures after being exposed for 7 days to RA+- NPs, RA--NPs, or conditioned medium (CM) obtained from the centrifugation of a nanoparticle suspension left in culture for 7 days. Data are expressed as mean ± SEM (n=5-15 coverslips) . ***P<0.001 using unpaired Student's t-test for comparison with SVZ control cultures. C) Percentage of NeuN-immunostained neurons in SVZ cultures after being exposed for 7 days to RA+- NPs or free-RA (solubilized in DMSO and added to the culture medium at different concentrations) . Data are expressed as mean ± SEM (n=6-15 coverslips) .

Figure 6 shows how the RA⁺-NPs induce differentiation of SVZ cells into functional neurons. A) Representative SCCI response profiles of 20 cells in a control culture and in a culture treated with RA⁺-NPs (0.1 pg/mL) . Graph shows percentages of neuronal-like responding cells (Hist/KCl below 0.8) in SVZ control cultures and in cultures exposed to RA+-NPs (0.01 or 0.1 g/mL) for 7 days. Data are expressed as mean ± SEM (n=6-9 coverslips). ***P<0.001 using Dunnet ' s Multiple Comparison Test. B) Graph depicts the percentages relative to control of NR1 protein expression normalized to GAPDH in cultures exposed to RA+-NPs (0.1 pg/mL) for 7 days. Below the graph, a representative western blot for 120 kDa NR1 and 37 kDa GAPDH expression is shown. The data is expressed as percentage of control ± SEM (n=5). *P<0.05 using paired Student's t-test for comparison with SVZ control cultures. Scale bars are 10 μιη. Figure 7 relates to the UV spectral scan of RA, RA conjugated to PEI, RA conjugated to PEI and after addition of DS, and RA conjugated to PEI and after addition of DS and zinc sulfate. The conjugate of RA with PEI was obtained by adding RA (0.3 mL; 2% w/v, in DMSO) dropwise to 6 mL of an aqueous solution of PEI (1% w/v, in pH 8.0 borate buffer). The suspension was centrifuged, the pellet ressuspended in PBS and characterized by spectrophotometry. In a separate experiment, DS or DS + zinc sulfate was added to the complex PEI:RA according to the methodology described in the Materials and Methods section, the suspension centrifuged and the pellet ressuspended in PBS and characterized by spectrophotometry. RA was dissolved in DMSO and diluted in PBS.

Figure 8 relates to the typical profile obtained by DLS for the formulation RA+-NPs (0.2). Results are expressed as number-weight average diameter.

Figure 9 relates to the proliferation of SVZ cells cultured in (A) proliferative (in serum free medium with growth factors as free floating neurospheres ) or (B) differentiation (after adherence on poly-D-lysine and in absence of growth factors) conditions. Data are expressed as mean ± SEM (n=4-6 coverslips) . SUMMARY OF THE INVENTION

The present invention is related with a novel strategy to modulate the activity and differentiation state of a cell by using nanoparticles able to release intracellularly hydrophobic drugs, including retinoic acid and other retinoid drugs. The inventors demonstrate that the internalization of the nanoparticles has a a minimal effect on cell viability and proliferation but a large impact on differentiation. Importantly, nanoparticle-conditioned medium is unable to promote the differentiation of stem cells indicating that neuronal differentiation is only mediated by internalization of the nanoparticles.

DETAILED DESCRIPION OF THE INVENTION

It is a first object of the invention a non-leaching nanoparticle formulation for the intracellular delivery of hydrophobic drugs comprising a hydrophobic drug physically immobilized to the nanoparticle, wherein the nanoparticle comprises: - -

a) a cationic polyelectrolyte selected from the group consisting of poly (ethyleneimine) , chitosan or poly ( lysine) ; b) an anionic polyelectrolyte selected from the group consisting of dextran sulfate, poly ( aspartic acid), hyaluronic acid or poly(acrylic acid).

In an embodiment of the invention, the drug is first complexed to one of the polyelectrolytes of the nanoparticle and then the resulting conjugate added to a second polyelectrolyte.

Normally, the nanoparticle formulation is physically immobilized to the nanoparticle.

Preferably, the hydrophobic drug is a retinoid drug.

In a more preferred embodiment of the invention the retinoid drug is retinoic acid.

Usually, the nanoparticle formulation has a pH sensitivity between pH 4 and pH 8.

"Non-leaching" nanoparticle formulation can be defined as a formulation that does not release enough encapsulated drug out of the cell (i.e., before the nanoparticle being internalized by the cell) in order to have biological activity. In practice, for example, the medium incubated with nanoparticles (10 μg/mL) for 7 days is unable to improve the neuronal differentiation of SVZ cells. However, the nanoparticles exposed directly to cells contribute for SVZ cell differentiation into the neural lineage. Therefore, in the present invention, the nanoparticle formulation is "non-leaching". _

It is a second object of the invention a process for preparing the nanoparticle formulation of the invention comprising the steps of:

complexing the hydrophobic drug to a negative or positive polyelectrolyte; and assembly of the polyelectrolyte- drug with a polyelectrolyte with an opposite charge.

The hydrophobic drug is immobilized, preferably, to a nanoparticle in a solvent formed by essentially by water.

It is a third object of the invention the nanoparticle formulation of the invention for using as an inductive agent to modulate cell activity. It is a fourth object of the invention the nanoparticle formulation of the invention for using as an inductive agent to differentiate cells at different stages of development or potential. It is a fifth object of the invention a method to modulate the activity and differentiation of cells, comprising the treatment of isolated cells with the nanoparticule formulation of the invention. In an embodiment of the invention the cell is a stem cell. Stem cell is defined as a cell that have both the capacity to self-renew (make more stem cells by cell division) as well as to differentiate int mature, speciallized cells. In another embodiment of the invention the cell is a progenitor cell. As defined by the International Society for Stem Cell Research, a progenitor cell is an early descendant of a stem cell that can only differentiate, but it cannot renew itself anymore. In contrast, a stem cell can renew itself (make more - - stem cells by cell division) or it can differentiate (divide and with each cell division evolve more and more into different types of cells) . A progenitor cell is often more limited in the kinds of cells it can become than a stem cell. In scientific terms, it is said that progenitor cells are more differentiated than stem cells .

In a more preferred embodiment of the invention the stem cells are neural stem cells. As defined by the International Society for Stem Cell Research, a neural stem cell is a type of stem cell that resides in the brain, which can make new nerve cells (called neurons) and other cells that support nerve cells (called glia) . In the adult, neural stem cells can be found in very specific and very small areas of the brain where replacement of nerve cells is seen. One of the specific locations of neural stem cells is at the subventricular zone (SVZ) .

The stem cell differentiation is achieved by internalization of the nanoparticle formulation.

Nanoparticles were prepared by complex coacervation, i.e., through the electrostatic interaction of polyethylenimine (PEI, polycation) and dextran sulfate (DS, polyanion) (Figure 1A) . Initially, complexes of RA with PEI were formed by the electrostatic interactions of the carboxyl groups of RA with the amine groups of PEI. Maximum loading is obtained for a ratio of amino groups to carboxylic acid groups of about 2: I⁹. In this work we used a ratio between 4:1 (for the NP formulation DS/PEI (5.0), considering the primary amines present in PEI) and 17:1 (for the NP formulation DS/PEI (0.2). The electrostatic interaction between RA and PEI is confirmed by a shift in the RA peak from 350 nm to 300 nm, as confirmed by spectrophotometry (Figure 7) . These complexes formed nanoparticles with 100 to 250 - - nm in diameter, although they tend to stick together and are not easy to resuspend. To stabilize the nanoparticle formulation, DS and zinc sulfate were added in successive steps¹⁶. The physical crosslinking of DS with PEI has been confirmed previously by Fourier Transform Infrared (FTIR) analysis¹⁶. Nanoparticles without zinc sulfate tend to aggregate and form large particles. Mannitol was added to the NP suspension during centrifugation steps and lyophilization to prevent their aggregation. Three different weight ratios of DS relatively to PEI were selected for our initial screening: 5.0 (large excess of DS) , 1.0 (similar weight of both polymers), and 0.2 (large excess of PEI). In each formulation we determined particle size, zeta potential and RA loading efficiency. A Brookhaven ZetaPALS analyzer was used to evaluate NP zeta potential and size distribution (deionized water) . The number-weighting profile of all formulations tested was usually unimodal (>99%) (Figure 8). The formulation DS:PEI weight ratio of 5 yielded nanoparticles with an average diameter between 40 and 120 nm, and a negative net charge, while the formulation DS:PEI weight ratio of 1 yielded large aggregates of nanoparticles (diameter > 500 nm) , with a positive net charge (Table 1) . This is in line with other results previously reported, indicating that an excess of one of the NP components is needed to act as a colloidal protective agent and preventing the coalescence of the NPs¹⁶. Nanoparticles prepared from the formulation DS:PEI weight ratio of 0.2 had an average diameter between 80 and 90 nm; however, presenting a significantly higher RA loading efficiency (48 versus 2%) and positive net charge (15 versus 2 mV) than the other formulations tested. The high loading efficiency is explained by the high content of PEI able to interact with RA. Based on the loading capacity and net charge results, nanoparticles obtained from the formulation 0.2 were selected for subsequent experiments. The morphology of these nanoparticles was then characterized by - - transmission electron microscopy (TEM) . TEM micrographs (Figure IB) show a similar range of sizes as observed by DLS .

The dissolution profile of nanoparticles is affected by pH . The dissolution of the nanoparticles (~10 mg/mL) was evaluated at pH 7.4, 6.0 and 5.0, under agitation, at 37°C. This pH range is typically found at the cytoplasm and intracellular organelles¹³. After 7 days, the suspensions were centrifuged, lyophilized and finally weighted. Interestingly, the disassembly of RA-containing nanoparticles (RA⁺-NPs) at pH 6.0 and 7.4 was lower than blank nanoparticles (RA^~-NPs) (Figure 1C) . This might reflect differences in the crosslinking of both preparations. RA might act as a crosslinker agent, where the carboxyl group interacts electrostatically with the amine groups of PEI, and the aromatic group interacts hydrophobically with the aromatic group of another RA molecule. RA⁺-NPs showed a mass loss of 15.4 ± 5.9%, 42 ± 3.8% and 54.5 ± 6.3%, at pHs 7.4, 5.0 and 6.0, respectively, over 7 days (Figure 1C) . Our results indicate that acidic pH enhanced the degradation of the nanoparticles likely due to the protonation of PEI amine groups (in excess relatively to the sulfate groups in DS) and the concomitant repulsion between positive charges¹⁷.

The release of RA from nanoparticles is dependent on the external pH. Polyelectrolyte complexes change their three- dimensional conformation according to the pH, affecting the release of RA. To evaluate this effect, nanoparticles were incubated at pH 7.4, 6.0 or pH 5.0, at 37°C, under agitation. At determined times the nanoparticle suspensions were centrifuged and the supernatant evaluated by spectrophotometry at 350 nm. The nanoparticle diameter was also evaluated at each time point (Figure 2A) . At pH 7.4, the initial diameter (-100 nm) is roughly maintained over 15 days, decreasing afterwards to approximately 60 nm. At pH 6.0 and 5.0, higher aggregation was - - observed for all the time points; however the population with diameters between 100 and 1000 nm (number-weighting average) accounted for most (> 95%) of the nanoparticles in suspension (Figure 2A) .

Taking into account that RA solubility is approximately 63 ng per mL at physiological pH⁸, most of RA released at pH 7.4 is complexed with PEI (Figure 2B) . Indeed, the complexation of RA to PEI is confirmed by a shift in the RA peak at 350 nm, which is not affected by the subsequent addition of DS (Figure 7) . Interestingly, the release of RA at pH 5.0 or 6.0 is much lower than pH 7.4, over the same period of time. This can be due to the crystallization of the RA and the aggregation of the nanoparticles at acidic pHs which decreases RA diffusion from the nanoparticles ¹⁸' ¹⁹. Alternatively, RA is released by the nanoparticles at higher percentages than the values assessed, and the low values observed by us is because RA precipitates at acidic pHs circumventing its real quantification .

RA⁺-NPs could potentially be used to induce neurogenesis and influence the brain regenerative capacities. In adult mammalian brain, neurogenesis persists in restricted neurogenic niches. These resources play a central role in the generation and integration of new neurons into pre-existing neural circuitry and are crucial for the maintenance of brain integrity and plasticity^20-22. The SVZ is the main neurogenic niche of the adult rodent brain^20-22 and contains a population of neural stem cells that can give rise to neurons, astrocytes, and oligodendrocytes. Therefore, SVZ cells have a huge potential for stem cell-based brain repair strategies. Previous studies have shown that nanoparticles (typically below 100 nm) formed by PEI (polycation) complexed with DNA (polyanion) can be internalized by adult mammalian brain cells^23-25. However, in spite of the - - importance of SVZ neural stem cells for brain physiology and repair, there is no consistent information concerning nanoparticle uptake and intracellular drug release in SVZ cells. To evaluate the kinetics of nanoparticle uptake by SVZ cells, they were exposed to fluorescein isothiocyanate (FITC)- conjugated nanoparticles and their uptake assessed by confocal live cell imaging and immunocytochemistry . RA⁺NPs suspended in cell medium showed a diameter of 250 nm and a zeta potential of -22.4 ± 4.4 mV. The negative zeta potential indicates a rapid electrostatic interaction with proteins and other elements from the culture medium. One hour after cell treatment, nanoparticles accumulate rapidly in cell cytoplasm (data not shown) . This accumulation becomes even more expressive at 5 h (Figure 3A) . This is in line with other studies, showing that PEI-based nanoparticles can escape rapidly endosomal fate due to their buffering capacity leading to osmotic swelling and rupture of endosomes¹⁵. At 18 h after cell treatment, the FITC-conjugated nanoparticles are distributed overall the cell cytoplasm (Figure 3B) .

To determine the cytotoxicity of these nanoparticles, SVZ neurospheres were exposed to RA⁺-NPs or RA^~-NPs for 2 days and cell death, presumably involving apoptosis or necrosis, was then evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidium iodide (PI) staining, respectively. No significant effect on cell apoptosis was observed for all the experimental groups tested with nanoparticle concentrations up to 100 μg/mL (Figure 4A) . In contrast, cell necrosis increases significantly for nanoparticle concentrations of 100 μg/mL (~4-fold) (77.3 ± 9.1%; n=4 coverslips; 2938 cells counted; P<0.0001), relatively to the control (17.8 ± 1.3%; n=9 coverslips; 8618 cells counted) (Figure 4B) . The toxic effects of 100 μg/mL RA⁺-NPs was not due - - to RA since the void formulation RA^~-NPs was equally toxic at the same concentration (Figure 4B) . Based on these results, RA⁺-NPs are not cytotoxic for concentrations equal or below to 10 μ$/τη1,. Moreover, at these concentrations, the internalization of the nanoparticles did not affect significantly the morphology of the cells. Untreated cells or cells treated with RA⁺-NPs (1 μς/πιΐ,) for 7 days were characterized by immunocytochemistry against neuron-specific class III beta-tubulin (Tujl) and glial fibrillary acidic protein (GFAP) . RA⁺-NPs-treated cells present a healthy morphology undistinguishable from untreated cells (Figure 4C) .

To investigate the effect of RA⁺-NPs on cell proliferation, SVZ neurospheres were exposed to a wide range of nanoparticle concentrations (0.001 to 10 pg/mL) for 2 days and then exposed to bromodeoxyuridine (BrdU) in the last 4 h of the treatment. BrdU is a synthetic nucleoside that can be incorporated into newly synthesized DNA substituting for thymidine during cell replication. SVZ cells either when grown under proliferative (in serum free medium with growth factors as free floating neurospheres) or differentiation (after adherence on poly-D-lysine and in absence of growth factors) conditions exposed to RA⁺-NPs had similar proliferation rates as control cells not exposed to nanoparticles (Figure 9) .

SVZ cell cultures are mixed cultures of immature cells, neurons, astrocytes, oligodendrocytes, neuronal and glial progenitors in different stages of differentiation^26-28. In untreated SVZ cultures we detected about 5 to 15% of new neurons (detected by SCCI and NeuN immunocytochemistry; Figure 5) , which is in accordance with the percentage of neurons obtained from standard methods of isolating neural stem cells in vitro²⁸' ²⁹. To evaluate the effect of RA⁺-NPs on neuronal differentiation of SVZ cells, the cells were cultured in differentiation conditions for - -

7 days in media containing the nanoparticles and then immunolabeled for NeuN, a neuronal-specific nuclear protein present on mature neurons^30-32. In the presence of RA⁺-NPs at concentrations of 0.1 pg/mL (19.6 ± 2.3%; n=5 coverslips; 3460 cells counted; P<0.001) and 1 μg/mL (19.2 ± 2.2%; n=7 coverslips; 4954 cells counted; P<0.001), a significant increase in the percentage of NeuN-positive cells was observed relatively to the untreated cells (11.8 ± 0.6%; n=13 coverslips; 7306 cells counted) (Figure 5A) . The proneurogenic effect mediated by RA⁺- NPs was absent in cell cultures treated with void nanoparticles (RA^~-NPs) , suggesting that this effect was due to the RA and not due to the nanoparticle formulation per se (Figure 5B) . Importantly, the neuronal differentiation effect was absent for high concentrations (> 1 ng/mL) of nanoparticles indicating that an optimal concentration window of RA exists to drive the neuronal commitment of SVZ cells. Most in vitro studies, using embryonic stem cells and neural stem cell cultures isolated from the SVZ and hippocampus, suggest that RA exposure stimulates neurogenesis and neuronal maturation^{7, 33-37}. However, the range of RA concentrations used in these studies ranged from 0.1 to 1 μΜ. These concentrations are 100 to 1000 fold higher than the payload of RA from nanoparticles that induces neurogenesis in our studies. Considering the RA payload per mg of NPs, we found that concentrations of RA payload below 4 nM RA (corresponding to 0.1 pg/mL nanoparticles) and above 40 nM RA (corresponding to 1 yg/mL nanoparticles) did not promote neuronal differentiation (Figure 5C) . This is consistent with the effect of RA in animal studies³⁸' ³⁹. The acute administration of RA (0.15 mg/kg) helped to reverse an age-related deficit in long-term potentiation amplitude and improved the ability of the mice to perform relational learning tasks³⁸. In contrast, the long-term systemic administration (1 mg/kg, for 42 days) of RA in mice leaded to a decrease in both proliferation and neuronal production in the hippocampus, which were accompanied by a significant reduction - - in the ability to perform a learning task . In this case, one of the RA effects was to suppress SVZ neurogenesis³⁹.

Because the differentiation process could be either due to RA release in the medium or within the cell, nanoparticles were left in serum free media (SFM) cell medium devoid of growth factors for 7 days, at 37 °C, then centrifuged and the resultant supernatant was collected, and used to treat SVZ neurospheres (7 days treatment protocol) . Interestingly, no significant proneurogenic effect was observed when the nanoparticle conditioned medium (CM) was added to SVZ cells (Figure 5B) . This shows that the differentiation of SVZ cells was only mediated by the internalization of the RA⁺-NPs and concomitant intracellular release of RA. We speculate that the spatial gradient of RA within the cell is likely the cause of this biological response; however future studies are needed to address completely this issue.

We also found that free RA solubilized in medium applying the same treatment protocol as for the NP, has only achieved a proneurogenic effect with a concentration of 10 μΜ (1000 fold higher concentration than RA released from nanoparticles) (148.6±22.9% percentage relative to control; n=6 coverslips from 3 independent cell cultures) (Figure 5C) . Similarly to what was observed with RA⁺-NPs, this neuronal differentiation effect was also absent for higher concentrations of free RA (50 μΜ) (81.3 ± 17.8% percentage relative to control; n=6 coverslips from 3 independent cell cultures). Therefore, RA signaling really depends on an optimal concentration window to drive the neuronal commitment. Importantly, results also stress that neuronal differentiation induced by RA⁺-NPs requires lower RA concentrations as compared with free RA. Therefore, our formulation is advantageous as comparing to the direct incubation with RA. To assess functional neuronal differentiation, we measured intracellular calcium ([Ca²⁺]i) variations in single SVZ cells, following KC1 depolarization and histamine stimulation²⁹. KC1 depolarization leads to the massive entry of Ca²⁺ through voltage-dependent calcium channels in differentiated neurons and is used as a functional marker of neuronal differentiation⁴⁰. Histamine stimulation increases [Ca²⁺]i in SVZ immature cells but not in neurons or in glial cells ²⁹. Therefore, mature neurons have low Hist/KCl ratios (below 0.8)²⁹. To evaluate [Ca²⁺]i variations, SVZ cells were loaded with the Fura-2AM calcium probe, perfused continuously for 15 min with Krebs solution, and subsequently stimulated for 2 min with 50 mM KC1 followed by 2 min stimulation with 100 mM histamine (Figure 6A) . In control cultures, most of the cells showed a predominant immature-like profile, characterized by an increase in [Ca²⁺] i in response to histamine but a negligible response to KC1 depolarization. In contrast, SVZ cells treated with RA⁺-NPs at concentration of 0.1 g/mL displayed an increase in the [Ca ⁺]i in response to KC1 but not to histamine stimulation, indicating their neuronal-like state (Ctrl: 4.8 ± 1.9%, 9 coverslips, 838 cells; 0.1 g/mL: 28.2 ± 5.2%, 8 coverslips, 1117 cells; P<0.001) (Figure 6A) . Moreover, these cells were characterized for the expression of NMDA subunit type 1 (NRl) by western blot analysis. Functional NMDA receptors form the molecular basis of synaptic plasticity and depend on the presence of the channel-forming NRl subunit⁴¹. In line with the previous results, SVZ cultures that presented a neuronal-like profile (treated with 0.1 pg/mL of RA⁺-NPs) also show a higher expression of NRl (-150% comparing with control cultures) (Figure 6B) .

We report a novel method to modulate the differentiation of SVZ cells into neurons involving the use of RA⁺-NPs. We show that our NP formulation is very effective in - - loading RA (86 ± 28 μq of RA per mg of NP) , a small molecule with low aqueous solubility, and to release RA at concentrations higher than its solubility limit (> 63 ng/mL, at physiological pH⁸) due to its complexation with PEI . We further show that the RA⁺-NPs have approximately 200 nm in diameter, positive net charge, and disassemble preferentially at acidic pHs . These RA⁺- NPs can be taken up rapidly by SVZ cells (first 5 h) under the tested conditions and localize in the overall cell cytoplasm after 18 h. The NPs are not cytotoxic and do not interfere with cell morphology and proliferation for concentrations below 100 μg/mL. Importantly, our results show that the intracellular internalization of RA⁺-NPs contribute for neurogenesis and therefore highlighting the importance of drug spatial positioning and concentration in terms of stem cell differentiation. We anticipate that these nanoparticles might be delivered into the brain via the nasal cavity, as recently demonstrated for the delivery of cells⁴². Thus, nanoparticle- mediated delivery of neurogenic-inductive factors (proteins, DNA, siRNA, mRNA) ⁴³ might provide a new opportunity for the treatment of various neurodegenerative disorders.

EXPERIMENTAL PART Materials

All-trans retinoic acid, polyethylenimine (PEI, average Mw ~25 kDa by LS, average Mn -10 kDa by GPC, branched) , zinc sulfate heptahydrate (ZnS0₄) ninhydrin reagent solution, Azure-A, fluorescein isothiocyanate (FITC) and osmium tetroxide were purchased from Sigma-Aldrich . Dextran sulfate (DS, Mw=500 kDa) was purchased from Fluka. Dimethyl sulfoxide (DMSO) and Ethanol (EtOH) were acquired from Merck. All reagents were used without further purification. - -

Examples

Example 1 Fluorescent labeling of PEI

PEI (10 g) was dissolved in 90 mL of borate buffer (pH 8.5) and a 10-fold molar excess of FITC, dissolved in 2 mL of DMF, was added dropwise and stirred for 1 hour. The solution was transferred to a dialysis membrane with 6 - 8 kDa cut-off and dialyzed against milliQ water. The dialyzed solution was freeze- dried for 2 to 3 days, protected from light.

Example 2

Preparation of nanoparticles

Nanoparticles with a weight ratio of DS to PEI of 1 were prepared by adding dropwise 0.6 mL of RA (2% w/v, in DMSO) ) into 12 mL of PEI (1% w/v, in pH 8.0 borate buffer). The formation of complexes between RA^~PEI occurred immediately and was allowed to proceed for 30 min with intense magnetic stirring. Then, 12 mL of DS solution (1% w/v) was added dropwise and stirred for another 5 min. Finally, 1.2 mL of ZnS0₄ aqueous solution (1M) was added and stirred for 30 min. Nanoparticles were centrifuged 3 times in 5% mannitol solution at 14,000 g for 20 minutes. Supernatants from each step were collected to determine PEI and DS amounts in nanoparticles. Resulting nanoparticles were frozen and lyophilized for 4 days to obtain a dry powder. Lyophilized nanoparticles were stored at 4°C. Similar protocol was adopted for the preparation of nanoparticles with a weight ratio of PEI to DS of 5 and 0.2, by changing the concentration of the polymers accordingly. Blank nanoparticles were prepared using the same procedure in the absence of RA.

Example 3

Characterization of the nanoparticles

The morphology of the nanoparticles was evaluated by transmission electron microscopy (TEM) . The suspension of nanoparticles (5 mg/mL) was stained with osmium tetroxide solution (1%, w/v) and single drops of the solution dropped on a TEM mesh copper grid coated with Formvar. The nanoparticles were then observed by TEM on a JEOL JEM-100 SX microscope at 80 kV.

Particle size was determined using light scattering via Zeta PALS Zeta Potential Analyzer and ZetaPlus Particle Sizing Software, v. 2.27 (Brookhaven Instruments Corporation). Nanoparticles suspended in water and sonicated for short times (< 10 min) were used. Typically, all sizing measurements were performed at 25°C, and all data were recorded at 90°, with an equilibration time of 5 min and individual run times of 60 s (5 runs per measurement). The average diameters described in this work were obtained from plots displaying the number of nanoparticles per diameter. The zeta potential of nanoparticles was determined in a 1 mM KC1 pH 6 solution, at 25°C. All data were recorded with at least 6 runs with a relative residual value (measure of data fit quality) of 0.03.

Example 4

Determination of weight ratio of cationic to anionic polymer in nanoparticles (Azure-A and Ninhydrin Tests)

Anionic-to-cationic polymer (DS:PEI) mass ratios in nanoparticles can be determined by subtracting the known amount - - of PEI and DS in the supernatant of completely centrifuged particles from the initial amount present. The amount of PEI was determined using ninhydrin. Twenty microliters of supernatant from a centrifuged nanoparticle suspension were mixed with 380 mL of an acetic acid solution (0.05% w/v) . One milliliter of ninhydrin reagent solution was then added. The mixture was placed into a boiling water bath for 10 min and then cooled to room temperature. Three milliliters of 95% ethanol were added and mixed. The amount of PEI was then determined spectrophotometrically at 575 nm using a calibration curve from 10 to 30 mg/ml of PEI.

The amount of unreac ed DS in the supernatant was detected spectrophotometrically at 630 nm using an azure A dye binding method in which 400 mL of supernatant from a centrifuged nanoparticle suspension were mixed with 1.0 mL of azure A (10 mg/mL) . Again a calibration curve of 1.0 to 3.5 mg/mL of DS was used to determine the concentration of unknown samples. Example 5

Loading efficiency of RA in nanoparticles

In order to determine the amount of RA in loaded nanoparticles, a given amount of nanoparticle powder was dissolved in 1 mL of DMS0:HC1 (9:1) solution and the absorption for RA at 350 nm was measured. The RA concentration was obtained from a calibration curve from 0.47-0.0023 mg/mL of RA in DMS0:HC1 (9:1) solution.

Loading efficiency was calculated using the following equation : - -

. Residual amount of RA in the nanoparticles _{1 ΛΛ}

Loading Efficiency = p— - :— ΐτπ— ^: x 100

^{& J} Feeding amount of RA

Example 6 RA release from nanoparticles

Nanoparticles (2.5 mg) were placed in PBS (0.5 mL) and incubated under mild agitation, at 37°C. At specific intervals of time, the nanoparticle suspension was centrifuged (at 20,000 rpm for 20 min) and 0.4 mL of the release medium removed and replaced by a new one. The reserved supernatant was stored at 5°C until the RA content in release samples was assessed by spectrophotometry at 350 nra . Concentrations of RA were determined by comparison to a standard curve. All analyses were conducted in triplicate.

Example 7

In vitro degradation of polymeric nanoparticles.

Nanoparticles with or without RA (5 mg) were immersed in 10 mM PBS (0.5 mL, pH 7.4) and citrate buffer pH 5 and 6 and incubated at 37 °C, under mild agitation. In one experimental set, the incubation lasted 7 days, after which, the nanoparticle suspension was centrifuged (20,000 g for 15 min) and the pellets lyophilized for final mass and total RA assessment. On the second experimental set, the incubation lasted for 21 days and at daily intervals, the nanoparticles suspension was centrifuged (20,000 g for 15 min) and 0.4 mL of the release medium removed and replaced by a new one, for RA quantification and particles size measurements. At the final day, the resulting nanoparticle suspensions were lyophilized for 3 days to obtain a dry powder for mass determination. - -

Example 8

SVZ cell cultures All experiments were performed in accordance with NIH and European (86/609/EEC) guidelines for the care and use of laboratory animals. SVZ cells were prepared from 1-3 day-old C57BL/6 donor mice as described by Agasse et al . (2004). Briefly, mice were killed by decapitation and the brains were removed and placed in calcium- and magnesium-free HBSS solution (Gibco, Rockville, MD, USA), under sterile conditions. Fragments of SVZ were dissected out of 450 m-thick coronal brain sections using a Mcllwain tissue chopper and then SVZ was digested in 0.025% trypsin (Gibco) and 0.265 mM EDTA (Gibco) (10 in, 37°C), following mechanical dissociation with a P1000 pipette. The cell suspension was diluted in serum-free medium (SFM) composed of Dulbecco's modified eagle medium [ ( DMEM) /F12 + GlutaMAXTM-I ) ] supplemented with 100 U^ml penicillin, 100 g-^ml streptomycin, 1% B27 supplement, 10 ng-rnl epidermal growth factor (EGF) and 10 ng^ml basic fibroblast growth factor (FGF)-2 (all from Gibco). Single cells were then plated on uncoated Petri dishes at a density of 3000 cells/cm² and were allowed to develop in an incubator with 5% C0₂ and 95% atmospheric air at 37°C. Six-day-old neurospheres were adhered for 48hrs onto

24 well plate poly-D-lysine-coated glass coverslips for all experiments except western blots, which were left adherent onto poly-D-lysine-coated 6well plates, all in SFM devoid of growth factors. Then, the neurospheres were allowed to develop for 2 or 7 days at 37°C in the presence a broad range of RA^'releasing NP concentrations. Cell death assays were done after 2 days of incubation. At the end of the 7 day treatment, Single Cell Calcium Imaging (SCCI) experiments, western blots and immunocytochemistry were performed. - -

Example 9

Single Cell Calcium Imaging To functionally characterize neuronal differentiation in SVZ cells, variations of intracellular calcium concentration ([Ca²⁺]i) following stimulation with 50mM KCl and ΙΟΟμΜ histamine ( Sigma-Aldrich) were analyzed. KCl depolarization causes the increase of [Ca²⁺]i in neurons, whereas stimulation with histamine increases [Ca²⁺]i in stem/progenitor cells. SVZ cultures were loaded for 40min at 37 °C with 5μΜ Fura-2 AM (Molecular Probes), 0.1% fatty acid-free bovine serum albumin (BSA) , and 0.02% pluronic acid F-127, in Krebs buffer (132 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM HEPES, pH 7.4). After a lOmin post-loading period at room temperature (RT) , the coverslip was mounted on RC-20 chamber in a PH3 platform (Warner Instruments) on the stage of an inverted Axiovert 200 fluorescence microscope (Carl Zeiss) . Cells (approximately 100 cells per field) were continuously perfused with Krebs and stimulated by applying ΙΟΟμΜ histamine or high- potassium Krebs solution (containing 50mM KCl, isosmotic substitution with NaCl) by the mean of a fast pressurized (95% air, 5% C0₂ atmosphere) system (AutoMate Scientific Inc.). [Ca²⁺]i was evaluated by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation (750 milliseconds) at 340 and 380 nm, using a Lambda DG4 apparatus (Sutter Instrument) and a 510 nm band-pass filter (Carl Zeiss) before fluorescence acquisition with a 40x objective and a CoolS AP digital camera (Roper Scientific) . Acquired values were processed using the MetaFluor . software (Universal Imaging Corporation) . Histamine/KCl values for Fura-2 ratio were calculated to determine the extent of neuronal maturation in cultures. The percentage of cells displaying a neuronal-like profile was calculated on the basis of the histamine (Hist) /KCl ratio. Example 10

Internalization studies

In order to visualize the NPs internalization, cells were incubated overnight with the RA^~releasing NPs (10 g/mL) and then fixed in methanol-acetone 1:1 for 20 min at -20°C. After fixation, non-specific binding sites were blocked with 6% BSA (Sigma-Aldrich) in PBS for Ih. Cells were subsequently incubated with the primary antibody mouse monoclonal anti-FITC (1:100, Sigma) in PBS containing 0.3% BSA and 0.1% Triton X-100 (Sigma). Thereafter, coverslips were rinsed in PBS and incubated for lh RT with the secondary goat anti-mouse Alexa 594 (1:200, Molecular Probes). After rinsing with PBS, cell preparations were incubated with Hoechst 33342 (2pg/ml, Molecular Probes) in PBS 5min RT for nuclear staining. Finally, the preparations were mounted using Dako Fluorescent Mounting Medium (Dako North America Inc.). Fluorescent images were acquired using a confocal microscope (LSM 510 Meta, Carl Zeiss, Gottingen, Germany) .

Example 11 Western Blots

Cells were washed with PBS and incubated in a lysis buffer (137:mM NaCl, 20mM Tris-HCl, 1% Triton X-100, 10% glycerol, ImM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 yg/ml leupeptin, 0.5 rtiM sodium vanadate (all from Sigma), pH 8). Total protein concentration from the lysates was determined by BCA assay. Then, proteins were resolved in 15% SDS polyacrilamide gels and then transferred to PVDF membranes with 0.45 pm pore size in the following conditions: 300mA, 90min at 4°C in a solution containing lOmM CAPS and 10% methanol, pH 11) (protocol adapted from Pinheiro et al. (2005)). Membranes were _ - blocked in Tris-buffer saline containing 5% low-fat milk and 0.1% Tween® 20 (Sigma) for one hour, at RT, and then incubated overnight at 4°C with the primary antibody solution diluted in 1% TBS-Tween, 0.5% low fat milk. The following primary antibodies were used: rabbit monoclonal anti-NRl 1:100 (CHEMICON International Inc.). After rinsing three times with TBS-T 0.5% low-fat milk, membranes were incubated for lh at RT with an alkaline phosphatase-linked secondary antibody, anti-rabbit IgG 1: 20 000 in 1% TBS-T 0.5% low-fat milk (GE Healthcare UK Limited Little Chalfont, Buckinghamshire, UK) . For endogenous control immnunolabelling, primary antibody solutions consisted of mouse monoclonal anti-GAPDH 1:10 000 (Millipore, MA, USA). Protein immunoreactive bands were visualized in a Versa-Doc Imaging System (Model 3000, BioRad Laboratories, CA, USA) , following incubation of the membrane with ECF reagent (GE Healthcare UK Limited Little Chalfont, Buckinghamshire, UK) for 5min .

Example 12

Immunocytochemistry

Cells were fixated with 4% paraformaldehyde (PFA) previously heated at 37°C for 30min and placed for 20min in permeabilizing solution (0.3% BSA (Sigma), 3% Triton X-100). After washing three times with PBS, unspecific binding was prevented by incubating cells in a 3% BSA and 0.3% Triton X-100 solution for 30min, at RT . Cells were kept overnight at 4°C in a primary antibody solution, then washed with PBS the following day, and incubated for lh RT with the corresponding secondary antibody. Antibodies were used as listed: mouse anti-Tujl (1:100); rabbit anti-GFAP (1:100) (Cell Signaling Tech.) in 0.1% Triton X-100, 0.3% BSA solution; Alexa Fluor 594 anti-mouse; Alexa Fluor 488 anti-rabbit (all 1:200 in PBS, from Molecular Probes, Oregon, USA) . For nuclear labelling, cell preparations - - were stained with Hoechst 33342 (2 μg/ml) (Molecular Probes, Eugene, Oregon, USA) in 0.3% BSA, for 5min RT and mounted in Dakocytomation fluorescent medium ( Dakocytomation Inc., California, USA) . Fluorescent images were acquired using a confocal microscope (LSM 510 Meta, Carl Zeiss, Gottingen, Germany) .

Table 1 Physico-chemical characteristics of DS/PEI nanoparticles either with or without RA

Ratio³ Emulsion^d . ^'If?, Rft payloacf

Formulation RV³

w/w % . ■ mV rett .rim ug/mg.

DS/PEI 5 1 -34.7+3.0 43.4 0.074 74 0.311 - -

..DS/PEI+RA .5 : 6.2 -42.618.1 ^{' '} 115.·, 0.283 67 -0.557 ^' 2.211.2 18.619.2

DS/PEI 1 n.a. n.a. 54 r 0.510i n.a. n.a. - -

DS/PEI +RA i ...7,3 : '.+1.9+0.5 . ri ii¹ .. 0.2281., n.a. n.a. ...0.4+0.2 ^' 2.5+1.5

DS/PEI 0.2 22.5 +14.912.8 91 0.044 61 0.078 _

: DS/PEIr+RA : ■ 0.2 . 34,12 +15; 6+1.4 ^"'".SO · .0.074 224 0.503 .48.3+15.9 .86.^"1+28.4 ^a - Polyelectrolyte initial weight ratio. The real ratio for the 0.2 and 0.2RA formulations was estimated to be 0.585 and 0.599, respectively.

b - Yield (without mannitol)

c - Zeta potential in mV

d - Peak diameter after the emulsion

e - Relative variance

¹ - anoparticles size after freeze-drying and ressuspended in aqueous buffer

9 - Loading efficiency

h - Amount of RA per mass of nanoparticle (without mannitol)

¹ - Formation of aggregates; values after removing the formed agglanerates.

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Claims

1. A non-leaching nanoparticle formulation for the intracellular delivery of hydrophobic drugs comprising a hydrophobic drug immobilized to the nanoparticle, wherein the nanoparticle comprises: a) a cationic polyelectrolyte selected from the group consisting of poly (ethyleneimine) , chitosan or poly ( lysine ) ; b) an anionic polyelectrolyte selected from the group consisting of dextran sulfate, poly ( aspartic acid), hyaluronic acid or poly(acrylic acid).

2. The nanoparticle formulation according to claim 1, wherein the drug is physically immobilized to the nanoparticle .

3· The nanoparticle formulation according to claim 1 or 2, wherein the hydrophobic drug is a retinoid drug.

4. A nanoparticle formulation according to claim 3, wherein the retinoid drug is retinoic acid.

5. A nanoparticle formulation according to any one of claims 1 to 4, having a pH sensitivity between pH 4 and pH 8.

6. A process for preparing the nanoparticle formulation as claimed in any one of claims 1 to 5 comprising the steps of:

7. The process according to claim 6 wherein the hydrophobic drug is immobilized to a nanoparticle in a solvent formed by essentially by water.

8. The nanoparticle formulation according to any one of the claims 1 to 5 for using as an inductive agent to modulate cell activity.

9. The nanoparticle formulation according to any one of the claims 1 to 5 for using as an inductive agent to differentiate cells at different stages of development or potential

10. A method of inducing stem cell differentiation, comprising the treatment of isolated cells with a nanoparticule formulation of any one of claims 1 to 5.

11. The method according to claim 10 wherein the stem cells are neural stem cells.

12. The method according to claim 11 wherein the neural stem cells are located at the subventricular zone (SVZ) .

13. The method according to anyone of claim 10 to 12 wherein the stem cell differentiation is achieved by internalization of the nanoparticle formulation.