method for its preparation and their use

The present invention refers to a method for preparing folate functionalized nanodiamond particles in which the nanodiamond particles are coupled with a folate linker. Such nano-particles are used in the field of biomedicine for selective delivery to active proliferated cells for their imaging and tracking in nano-scale range.

Nano-diamonds are imagining as new and promising cellular biomarkers with high potential. It is known from the prior art that nano-diamond particles have a suitable surface for biological activation and functionalization (J. Opitz, A. Pohl, J. Schreiber, M. Mkandawire, U. Krause-Buchhoiz, G. Roedel, W. Pompe, T. Gubarevich, V.

Lapina, Nanodiamonds - a new quantum dot material and its possible applications in biology, VDI-Berichte Nr 2027, 105-110, 2008 and M. Mkandawire, A. Pohl, T. Gubarevich, V. Lapina, D. Appelhans, G. Roedel, J. Schreiber, J. Opitz; Selective targeting of green fluorescent nanodiamond conjugates to mitochondria in HeLa Cells, Biophotonics 2, No. 10, 596-606 (2009), J. Opitz, M. Mkandawire, M. Sorge, N. Rose, M. Rudolph, P. Krueger, 1. Hannstein, V. Lapina, D. Appelhans, W. Pompe, J.

Schreiber, G. Roedel; Green fluorescent nanodiamond conjugates and their possible applications for biosensing, Proceedings of SPIE 7754-7768, 2010). The Application of nano-diamond particles as light scattering labels and luminescent optical marker has been reported in Yuen Yung Hui, Chia -Liang Cheng and Huan- Cheng Chang,„Nanodiamonds for optical bioimaging", J.Phys. D: Appl.Phys. V.43, N.37., p.4021.2010.

Moreover, it is known that the vitamin folic acid has been extensively investigated for targeting various cancer cells, including ovary, kidney, uterus, testis, brain, colon, lung, and myelocytic blood that overexpress folic acid receptors. The cell- membrane folate receptor is a potential molecular target for tumor selective drug delivery, including various folate-chelate conjugates for diagnostic imaging (US 7, 598, 335).

Though different applications for the use of nanodiamond particles as optical marker are known there is still a need for optical markers with improved long-term photostability.

It was therefore the object of the present invention to provide nanodiamond particles with a high photostability which are easy to prepare.

For solving this problem, the method for preparing folate functionalized

nanodiamond particles with the features of claim 1 and nanodiamond particles with the features of claim 11 are provided. In Claim 15, a use according to the present invention are described.

According to the present invention a method for preparing folate functionalized nanodiamond particles is provided comprising the following steps:

- providing nanodiamond particles,

- modifying the nanodiamond particles with carboxyi groups by oxidation or ozonization to increase the number of reactive groups at its surface,

- coupling at least one folate with a bifunctional linker which is linkable to the carbon groups of the nanodiamond surface and to the folate, wherein one functionality of the biofunctional linker is blocked by a protective group, and - reacting the modified folate with the modified nanodiamond particles.

It is preferred that the nanodiamond particles are provided by detonation of 2- methyl-l,3,5-trinitrobenzene (TNT) and l,3,5-trinitroper-hydro-l,3,5-triazine (RDX).

The Afunctional linker preferably and amine, more preferably selected from the group consisting of N-tert-butoxycarbonyl-l,6-hexane-diamine, tert-butyi N-(6- aminohexyl)-carbamate, tert-butyl N-(5-aminopentyl)-carbamate, tert-butyl N-(3- aminopropyi)-carbamate, tert-butyl N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)- carbamate, and its mixtures.

In a preferred embodiment the coupling of the at least one folate with the Afunctional linker is performed by the following steps: - providing a suspension of the at least one folate in an organic solvent, preferably selected from the group consisting of dimethylsulfoxide, pyridine, dimethyl formamide and its mixtures,

- adding the linker having a protective group to the solvent,

- eliminating the protective group, and

- purifying the modified folate. It is preferred that nanodiamond particles are ozonised by the following steps: providing a suspension of the nanodiamond particles in an alkaline water solution, preferably comprising sodium hydroxide, - feeding ozone into the solution, acidulation of the solution, preferably by adding hydrochloric acid, and isolating the modified nanodiamond particles by centrifugation. In a further preferred embodiment the nanodiamond particles are oxidised by liquid phase oxidation, preferably with organic acids, inorganic acids, potassium permanganate, manganese dioxide, chromium (VI) compounds, halogens and its compounds, dimethyl sulfoxide, and its mixtures, and/or by gas phase oxidation, pref- erably a mixture of 0 ₂ + N ₂ (4 -20% 0 ₂ ), C0 ₂ +K ₂ C0 ₃ , H ₂ 0 +K ₂ C0 ₃ and/or 0 ₃ .

It is further preferred that in step {d): the modified nanodiamond particles are activated with 1,1- carbonyldiimidazol, the activated nanodiamond particles are reacted with the modified folates in an organic solvent, and the folate functionalized nanodiamond particles are purified by centrifuga- tion.

The folate are preferably selected from the group consisting of folic acid, folic acid modified by l-ethyl-3-(3-dimethy!amino-propyl)-carboimide and N ^' -hydroxy- succinimide.

The nanodiamond particles can be provided as a polydispersion of single nanodiamond particles and its aggregates. It is further preferred that at least a part of the nanodiamond particles comprise optically active structures, preferably nitrogen vacancy centres in the core of diamond particles and various fluorescence species in the diamond shell, preferably admixtures of metals Cu, Ti, Cr, Si, Zn, AL, M n, Fe and their compounds as oxides, oxy- organic compounds and salts.

According to the present invention also nanodiamond particles with a functionalized surface comprising at least one folate are provided. Such particle is conjugated with the folates via a chemical bonding wherein the density of the folate groups is at least one group per particle. It is further preferred that the nanodiamortd particles comprise optically active structures which are located on the surface and/or within the core of the particles, preferably nitrogen vacancy centres in the core of diamond particles or various fluorescence species in the diamond shell, preferably admixtures of metals Cu, Ti, Cr, Si, Zn, AL, Mn, Fe and their compounds as oxides, oxy- organic compounds and salts or modified by fluorescence organic dyes on surface shell.

The nanodiamond particles are preferably polycrystalline and/or microcrystalline.

Nanodiamond particles according to the present invention can be used as fluorescent marker for cells, in particular tumor cells, or as fluorescent tracking marker in tumor cells and active-proliferated cells.

These particles can be used as a nanoscale biomarker for cancer or chronic inflammatory diseases or as tracking imaging agent, e.g. to visualize the process of cancer tumors.

The present invention uses raw of the unique features of nanodiamond particles, including the nanoscale sizing, large chemically active surface area to mass ratio, surface modification, long-term photo stability, and fluorescence blinking and bleaching.

The present invention uses the fluorescence of the nanodiamond particles conjugated with the folate. The visualization of the nanodiamond particles can be done by fluorescence provided from nanodiamond particles or their aggregates.

The selective imaging of cancer or inflammatory cells is realized by the folate func- tionalized nanodiamond particles. It is an advantage of the present invention that the long-term imaging and tracking incomp!ex cellular cancer or inflammatory envi- ronments can be improved due to the exceptionally photostability without photo- bleaching and photoblinking of the inventive nanodiamond particles. The own fluorescence properties of the nanodiamond particles enables the visualization of cancer cells and the folate enables the selective delivery of the particles to the cancer cells. The present invention is explained in more detail with the reference to the following examples and figures. However, it has to be noted that these are only specific embodiments which do not limit the invention as claimed. Fig. 1 shows fluorescence excitation spectra of ultradispurse diamonds (UDD)for different wavelengths.

It can be seen from the spectra that UDD water suspension reveals a number of fluorescent centers with different characteristics. Various position of λ max. fluores- cence from 360 up to 540 nm. The fluorescence excitation spectrum practically coincides for 'blue' and 'red' diamonds.

Fig. 2a and 2b show structural setup of the nanodiamond particles coupled with the folates. The synthesis of these structures is carried out in three stages:

1) . Connection of FA carboxyl group with a one amino group of the linker.

2) . Activation of carboxy! group of UDD by N-hydroxysuccinimide.

3) . Reaction of activated UDA with another group of the linker. Fig, 3 shows spectra of fluorescence excitation for folic acid, nanodiamond particles and nanodiamond particles coupled with folic acid.

It can be seen from spectra that obtained conjugate ND-FA has new own fluorescent centers with λ max. fluorescence excitation under 330 nm.

Example 1

Preparation of nanodiamonds to the synthesis of conjugate Ultradisperse diamonds (UDD) obtained by explosive synthesis are known to be elementary particles with the size of 4-6 nm and surrounded by the coating of various non-diamond forms of carbon. For bench mark of functionaltzation of UDD of folic acid we propose to use carboxyl group situated on the UDD particle. UDD cleaned by the method {method of back titration) contains one carboxyl group for 5000-6000 carbon units, that is, in our opinion, is not effective at conjugate synthesis. The increasing content of carboxyl groups can be made by additional oxidation of lower degrees of carbon as hydroxyl, carbonyl and isolated double bindings; and also thanks to hidden forms of carboxy! as complex ethers, amides, nitrides. Due to the last argument we have used the method of ozonolization of Sp ² hybridized atom of carbon of the UDD surface in water-alkaline medium. The use of sodium hydroxide as a medium allows one to oxidize the appearing ozonid till carboxyl group and hydrolyze complex-ether groups till carboxyl group. Further stage is aciduiation of

UDD suspension by the solution of hydrochloric acid to pH=2-6 with the following centrifugation and up to ten / water cleaning. The process described above allowed one to increase the content of carboxyl groups in 2-3 times. Example 2

Ozonization of UDD suspension.

Ozonization of UDD suspension with the concentration of 5 g per 1 liter of water was made on the set-up for obtaining ozone with the flow speed of 10-15 ml per second and content of ozone in gas mixture of 3-5 %. The ozone was obtained of air mixture, cleaned by series connection of Tischenko bottles: starting with the concentrated sulfuric acid and then with dry granulated caustic soda. To the suspension of UDD, 0.1 g of caustic soda was added and while mixing through the bubble tube there was passing the current of ozone for 2 hours {excess is more than in 300 times). Additional oxidation was made by adding 30 ml of 30% hydrogen peroxide and holing the mixture during 1 hour. Then 10% hydrochloric acid was added up to receiving acid reaction and it was also hold during 1 hour. The upper transparent layer was decanted carefully and lower one was sent to centrifuging with the following water washing for 8-10 times. The remains of water were eliminated by double washing by dioxane. After drying for 24 hours at room temperature, the content of carboxyl groups was determined by the method described above. The molecular weight (without sorptive water) was within the limits of 1300-1560 (depending of various tests). Example 3

Synthesis of the nanodiamond particles-folic acid complex has been made in 3 stages according to the following scheme: 1. Addition of spacer of N-Boc-1,6- diamtnohexan (where, N-Boc = tret- butoxycarbontl) to folic acid (FA). 2. Elimination of protection (N-Boc) from aminogroups. 3. Activation of carbonyl group in nanodiamond and joint of FA. 1. Synthesis of y-[tertbuty!-N-(aminohexyl)carbomate] of folic acid (I)

To the suspension of folic acid (0.805 g, 1.83 miliimo!e) in a mixture of waterless dimethy!sulfoxide(DMSO) (30 ml) and pyridine (15 ml), N-Boc-l,6-diaminohexane

(0.44 g; 2.02 millimole) and dicyclohexylcarbodiimide (DCC) (0.949 g, 4.6 millimole) was added and mixed on the magnetic mixer in the atmosphere of nitrogen for 18 hours at room temperature. Subsequently the reaction mixture was filtered, and anhydrous diethyl ether (0.5 liter) cooled up to 0°C was added to the filtrate while mixing. Deposited yellow crystals were filtered and washed with diethyl ether and then dried under vacuum. The product output is 0.5 g (95%). In the result of the reaction there has been obtained y-[tertbuty!-l\l-(aminohexyl)carbomate) of folic acid (FA-AMH - BOC, I). NMR spectrum of FA-AMH-BOC: NMR spectrum. IDvlP Ή, (,lWCO-d6): 011,49 (br s,

1 H), 7,82 (br t,lH, NH),7,65 (d,j =8.8Hz) 6,93 (Br t, 1 Η,ΝΗ), 6,74 (Br n 1 H, NH), 6,63 (d,j=8.8 HZ,2H), 4,49(d,j=5.6 Hz, 2H), 4.36-4.23 (m, 1 H), 3.01 (td, j=6.0,6.0 Hz, 2 H), 2.88 (d,j=6.4 Hz, 2H), 2.29-2.15 (m,2H), 1.98-1 .82 (m, 2H), 1 .41-1.16 (m, 8H), 1.36 (s, 9H).

2. Synthesis of y-(6-aminohexyl) of folic acid (II)

To the junction I (0.995g, 0.38 milltmole) trifluoroacetic acid (TFA) (8 ml) was added and mixed on the magnetic mixer at the room temperature for 2 hours. TFA was eliminated from reaction mixture at high vacuum on the rotary evaporator. The obtained sediment was dissolved in anhydrous dimethylformamide (DMFA). Anhydrous pyridine was added drop to the obtained solution by drop up to setting of yellow sediment. After filtration, the sediment was washed with ether and dried under vacuum. The product output is 0.84 g (99 %). In the result of the reaction y- (6-aminohexy!) of folic acid (FA-AMH) has been obtained.

NMR spectrum of FA-AMH-BOC: NMR spectrum Ή, (.D;MCO d6): 88.65 (s, 1 H), 7.96 (m, 1 H, NH), 7.84 (m, 1 H, NH), 7.73 (m, 2H), 7.66 (d, J = 7.6 Hz, 2H), 7.32 (br s, 1 H),6.64 (d, J = 7.6 Hz, 2H), 4.50 (s, 2H), 4.38-4.24 (m, 1 H), 3.04 (br td, 2H), 2.77 (m, 2H), 2.38 - 1.77 (m, 4H), 1.73 - 1.16 (m, 8H).

3. Synthesis of complex of nanodiamond-[y-(6-aminohexYl) folic acid] For elimination of residual moisture, powder of nanodiamonds (50 mg) was crushed thoroughly in agate mortar and placed into a round-bottom flask with the capacity of 50 mt. Subsequently, 30 ml of anhydrous toluene was added and left for 1 hour. The toluene was removed by distillation and the product was dried under vacuum. In the atmosphere of nitrogen, 30 ml of anhydrous dimethylsulfoxide and 25 mg of 1,1-carbonyldiimidazol were added into the dried nanodiamonds and mixed for 10 minutes on the magnetic stirrer. The junction I! (60 mg) in the mixture of anhydrous DMSO (4.0 ml) and pyridine (2.4 ml) was added to the reaction flask and mixed for 24 hours. After mixing for 24 hours, 80 ml of anhydrous diethyl ether cooled up to 0°C was added to the reaction mixture. Light-yellow sediment was centrifugated and washed consequently by diethyl ether, dichloromethane and water. DMSO was added to the dried sediment and for 10 minutes processed by ultrasound and centrifugated at 4 000 g. The sediment was marked as ND-FA complex and studied by spectrophotometry methods. The obtained ND-FA complex is fine-dispersed dry powder of grayish-yellow color with the characteristic spectral properties.