Novel hypericum coated nanoparticle, method and kit for diagnosis and prognosis of cancer

Abstract

The invention describes a novel nanoparticle coated with a Hypericum perforatum extract and a novel diagnostic kit in which such nanoparticle is used and isolates cancer cells, achieving the effective, accurate and efficient diagnosis and prognosis of different types of cancer, by use of samples from liquid biopsies.

Description

[0001] NOVEL HYPERICUM COATED NANOPARTICLE, METHOD AND KIT FOR DIAGNOSIS AND PROGNOSIS OF CANCER

[0002] The present invention relates to a novel nanoparticle and a novel diagnostic kit, in which the nanoparticle is used, for effective, accurate and efficient diagnosis and prognosis of different types of cancer. It also relates to a method for the detection and / or prognosis of cancer by use of the nanoparticle and / or the diagnostic kit.

[0003] Cancer remains one of the leading causes of mortality worldwide. Early detection and accurate prognosis of cancer are essential for effective treatment and improved patient survival.

[0004] Currently, the most common method for obtaining a definitive cancer diagnosis involves cytological examination or biopsy of samples from the suspected area. These procedures are invasive, require specialized personnel, and often cause discomfort to the patients. In addition, there exists the possibility of false-negative results either due to incorrect sampling — where the biopsy may fail to capture cancerous cells — or due to interpretive error by the pathologist assessing the samples.

[0005] Meanwhile, various techniques enable the highly accurate detection of circulating tumor cells (CTCs) and circulating free DNA (cfDNA) through liquid biopsies, such as blood or urine samples. Methods such as quantitative real-time PCR (qRT-PCR) and flow cytometry allow for the identification of cancer cells by targeting specific surface markers or cell-associated mRNA, respectively. Additionally, next-generation sequencing (NGS) is employed for the detection and comprehensive sequencing of cfDNA. While these techniques offer high sensitivity, they are also prone to false positives due to the detection of non-specific cancer antigens or background signals, and they require fully equipped laboratories, expensive consumables, and highly trained personnel. Additionally, the turnaround time from sample collection to results often exceeds one week.

[0006] In the specific context of the diagnosis of bladder cancer - the sixth most common cancer globally and rising in incidence, particularly in developed countries - the gold standard remains urine cytology. However, this method suffers from low sensitivity, particularly in detecting low-grade tumors, and is influenced by variables such as tumor grade, the number of samples collected, and the expertise of the cytopathologist. While specificity may reach up to 93%, specialized laboratory infrastructure and trained personnel are necessary, and diagnostic results are generally not available sooner than two days following sample collection.

[0007] The bladder is a hollow organ in the lower abdomen whose main purpose is to store urine received from the kidneys (via the ureter) until micturition. Specialized transitional epithelial cells lining the urinary bladder and urinary tract, known as urothelial cells, accommodate the volume of urine produced by flattening under pressure. The bladder is also lined with smooth muscle that can relax to accommodate greater volumes, as well as contract (under voluntary or reflex control) to expel urine down the urethra and out of the body. The urothelial cells lining the bladder and urinary tract are constantly exposed to environmental, potentially mutagenic agents that are filtered into the urine by the kidneys. Unsurprisingly, 90% of bladder cancer cases, especially those in the developed world, arise from these urothelial cells, mostly in the bladder but on rare occasions in the urinary tract as well. While localized forms of urothelial cancer carry an excellent prognosis, if the smooth muscle is invaded, survival rates drop significantly.

[0008] Bladder cancer is usually first suspected due to hematuria and further identified with a cystoscopy, a telescopic endoscopy of the bladder, transabdominal ultrasound, and / or computer tomography (CT) urography. As many as 7 out of every 10 cases of bladder cancer are detected in early stages, thus allowing for resection and improved survival. Non-muscle-invasive bladder cancers (NMIBC) are typically removed by transurethral resection. Alternatively, a cystoscopy plus biopsy procedure may be used for certain resections. Intravesical cytotoxic therapy may be added for high-risk cases. Meanwhile, for the 30% of patients who present with muscle-invasive bladder cancer (MIBC), neoadjuvant or adjuvant chemotherapy is considered the standard to lower the risk of recurrence, and radical cystectomy is the mainstay surgical treatment. External beam radiation may also be used. For the treatment of metastatic disease (which currently has only a 5% 5-year survival rate), platinum chemotherapy remains the standard, though novel immunotherapies, namely checkpoint inhibitors, are growing in popularity as treatment options in first- line and beyond.

[0009] Although survival rates have improved with earlier diagnosis, robotic surgical techniques and the introduction of immunotherapy, bladder cancer remains a significant and rising contributor to cancer burden worldwide, especially in developed nations. There is thus, a clear and unmet need for a diagnostic system that is non-invasive, cost- effective, rapid, and reliable, with high sensitivity and specificity for early-stage cancer detection. An ideal solution would also reduce reliance on highly specialized personnel and laboratory infrastructure.

[0010] The objective of this present invention is to provide cost-effective and user-friendly diagnostic and prognostic systems with high sensitivity and accuracy for cancer detection at any stage, early or advanced, that are also suitable for the follow up of cancer patients under treatment, thereby allowing for centralized laboratories and point-of-care applications.

[0011] This objective is solved by providing a novel nanoparticle and a diagnostic kit according to the present invention. Further advantageous details, aspects and further embodiments of the invention are represented by the embodiments of the dependent claims.

[0012] In the context of the present invention, the novel nanoparticle is based on functionalized nanoparticle and natural plant-derived compounds. In particular, the invention utilizes components derived from Hypericum perforatum (commonly known as St. John’s wort, or “valsamo”), which is known to contain hypericin — a bioactive molecule with known antimicrobial, anti-inflammatory, antidepressant, and antitumor activities. Hypericin, a natural photosensitizer, has demonstrated significant antineoplastic activity upon irradiation in both in vitro and in vivo settings and holds promise for photodynamic therapy applications. Despite its documented pharmacological properties, there has been no prior application of Hypericum perforatum or its active constituents in cancer diagnosis or prognosis.

[0013] The invention integrates this bioactive compound into a nanoparticle-based platform capable of detecting specific cancer biomarkers in biological fluids. The system is designed to operate with minimal infrastructure and personnel, allowing for faster and more accessible diagnostic results. The proposed kit may be configured for the detection of urothelial carcinomas, including bladder cancer, by targeting disease-specific markers present on CTCs or in cfDNA extracted from patient samples such as urine or blood.

[0014] By combining a high selectivity and bioactivity of Hypericum perforatum-derived agents with the structural and functional advantages of nanoparticles, this invention enables a new class of diagnostic systems suitable for both centralized laboratories and point-of- care applications.

[0015] Description of the invention

[0016] The above-mentioned need for an easy to handle, efficient and reliable test system for cancer detection has the inventors motivated to search for new alternatives in identifying and isolating cancer cells from liquid biological samples.

[0017] It is due to the continuous effort of the inventors that with the present invention they can provide not only a method and a corresponding diagnostic kit for isolating and simultaneously characterizing - in general - cancer cells from a liquid biological sample, but they also can intensify the characterization to even allow a prognostic statement regarding the personalized situation of a patient.

[0018] The core elements of the present invention are: i. A magnetic nanoparticle coated with a Hypericum derived extract, which in comparison to standard or commercially available Hypericum extracts comprises none or at least substantially decreased amounts of fatty substances, in other words it is a degreased Hypericum perforatum extract which is hereinafter identified as AK1223 extract and is used for coating known magnetic nanoparticles. ii. The configuration of a nitrocellulose strip suitable for the detection of very low amounts of proteins found in cancer cells at different stages. Using this strip, one will be able to tell if the individual under examination has cancer and if this cancer presents metastatic or invasive potential at the time of the test.

[0019] A method to produce such AK1223 herbal preparation is described by the steps of soaking dry Hypericum perforatum herbs for at least 10 minutes and up to 24 hours at room temperature. Suitable solvents are water and / or alcohols or mixtures thereof. According to one embodiment methanol or ethanol are used as solvent. Alternatively, hot or boiling water can be used as solvent.

[0020] Depending on the choice of solvent and its capacity to extract fatty acids or other fatty substances (alcohols work better than water) the degreasing step is optional, needs to be only a filtering through filter paper or should be performed in a separatory funnel to remove a greater percentage (up to 80%) of the fatty substances. HPLC comparisons of a standardized Hypericum solution and the AK1223 extract of the invention are used to perform a quality check on the remaining presence of fatty substances.

[0021] The supernatant of this degreased extract is then dried by evaporating the remaining solvent and / or water and producing thereby a AK1223 concentrate and eventually a AK1223 powder. The resulting AK1223 concentrate, or powder is then used for coating the magnetic nanoparticles according to well-known standard methods leading to AK1223 coated magnetic nanoparticles.

[0022] According to a further embodiment the magnetic nanoparticle according to the invention are prepared employing an even more intensive extraction protocol wherein solvent extraction is repeated one to three times. The solvent to be chosen in the extraction protocol is typically alcohol, preferably methanol or ethanol. Alternatively, the solvent can also be water or a mixture of alcohol or water, preferably a mixture of 20:80, 30:70, 40:60 or 50:50 of alcohol :water.

[0023] According to a further embodiment the drying of the extract to a concentrate or powder can be performed either at room temperature, at temperatures risen in a range of 40-60°C or under lyophilizing condition.

[0024] According to an alternative embodiment the magnetic nanoparticle according to the invention are prepared employing an aqueous extracted solution of Hypericum perforatum (AK1223) which is prepared by adding dry pulverized herb to boiling water and let it stay for at least 10 min, then after the solution had cooled the extract is infiltrated using standard filter paper and the extract is then processed in a lyophilization procedure to produce lyophilized AK1223 powder.

[0025] Although the coating of nanoparticles is well predescribed we hereinafter describe as Example 2 a suitable coating protocol.

[0026] According to the invention the magnetic nanoparticle coated with a degreased Hypericum perforatum extract (AK1223) are to be used in a method to capture, isolate and / or concentrate cancer cells from liquid biological samples. It had been shown that the AK1223 extract is suitable and capable to specifically recognize and enter to cancer cells, as shown in the Figures 1 -4. Binding to magnetic nanoparticles the AK1223 extract is then used to firstly allow in suitable buffers the cancer cells to bind to the nanoparticle and then pull and isolate the nanoparticle due to their magnetic characteristics out from a liquid biological sample. Cells that are bound to the AK1223-nanoparticles are an enrichment of cancerous cells, which are then isolated, and which can subsequently be further characterized.

[0027] One, such further characterization is the use of the diagnostic kit and according to the method as described hereinafter. However, also other diagnostic characterization can be used to further characterize the cancer cells isolated from a given biological sample, which can be blood, serum, urine, lymph, mucus, stool or any other tissue biopsy of a patient.

[0028] The diagnostic kit according to the present invention contains AK1223 coated magnetic nanoparticles, which are e.g. prepared by using an Hypericum perforatum extract (AK1223) extracted from dry herb by soaking the plant material in solvent each time for 24 hours at room temperature, followed by degreasing in a separatory funnel so that the extract is washed to remove the greater percentage of fatty substance and the supernatant is evaporated to dryness e.g. at room temperature.

[0029] The diagnostic kit for capturing cancer cells, so-called circulating tumor cells (CTCs), from liquid biological samples comprises one or several containers with the following and suitable dilution puffers:

[0030] - the magnetic nanoparticle coated with the degreased or aqueous solution of Hypericum perforatum extract, which are useful as a cancer cell capturing tool;

[0031] - an appropriate cell lysis buffer;

[0032] - a tube containing a.) one or several lyophilized monoclonal antibodies for the detection of tumor markers hereinafter identified as capturing antibody, b.) and at least one detecting antibody, preferably a rodent anti-human antibody, c.) at least one secondary gold labelled monoclonal preferably an anti-rodent antibody, and d.) gold labelled biotin;

[0033] - a nitrocellulose strip optionally being incorporated into a handling cassette and containing in different lines e.) one or several tumor-marker-specific antibodies used for the detection of the same tumor markers of the cancer cells isolated with the AK1223 coated magnetic nanoparticles, wherein these tumor-marker-specific antibodies bind to different epitopes compared to the antibodies in step a.), and wherein optionally, these tumor marker specific antibodies are also bound by the detecting antibody of step b.), and f.) streptavidin.

[0034] According to one embodiment the diagnostic kit comprises the magnetic AK1223 coated nanoparticle as a cancer cell capturing tool, an appropriate lysis buffer, preferably the so- called RIPA buffer. In this RIPA buffer, which is commonly used in biological research, particularly for protein extraction from mammalian cells, the cells captured from the liquid biological sample are lysed to separate proteins form different cellular compartments.

[0035] According to a further embodiment the kit and the corresponding method comprise an optional step of removing red cells with erythrocyte lysis buffer (ELB) before reaching the step of the RIPA Buffer. This optional step is highly recommended when the liquid biopsy material contains blood.

[0036] With the kit it is particularly easy to perform the method of firstly isolating and concentrating tumor cells from a biological sample and further performing the diagnostic characterization as described as method hereinafter.

[0037] The cell lysis extract, preferably the fraction which contains membrane bound tumor markers is added to a tube containing in a suitable dilution buffer monoclonal antibodies according to one embodiment specifically binding the tumor markers E cadherin, Vimentin, EPCAM and / or Uroplaktin2 (UPKII). These antibodies are hereinafter identified as detecting antibodies, as they bind to the tumor markers to be detected and captured.

[0038] According to other embodiments alternative tumor cell markers or alternative cell markers can be used, which then has the consequence that the selection of detecting antibodies is to be adjusted.

[0039] In the tube of the kit the detecting antibodies then bind, depending on their specificity, to their tumor marker if it is present. A second set of antibodies, which is preferably selected as a gold labelled monoclonal antibody, is then active in the tube to bind to the detecting antibody. Later this gold labelled antibody is intensified by gold labelled biotin.

[0040] Typically, if the detecting (marker-binding) antibody is a rabbit anti-human then the secondary gold labelled monoclonal antibody is anti-rabbit. The skilled person knows how to build such cascades of antibodies, which recognizes other antibodies and thus any suitable combination is possible and included.

[0041] Also, the amount or concentration of the antibodies employed in the kit is given as an exemplary amount only and can be adjusted depending on the selection and / or specificity of the detecting antibodies. In a standard kit according to the invention antibodies are provided in a concentration of about 1 pg.

[0042] After the given time of incubation in the tube of the kit the binding reactions are completed and the marker-binding detecting antibodies have found their marker antigen, and further the secondary gold labelled antibodies have found the detecting antibodies. Biotin included in the reaction will help to intensify a signal.

[0043] These cascades of antibodies are now transferred on to the nitrocellulose strip of the kit. This nitrocellulose strip is prepared with one or several capturing antibodies on defined positions in different tracks, which each bind one of the same tumor or cellular markers as the detecting antibodies in the tube of the kit. However, the capturing antibodies need to bind always to a different epitopes of tumor or cellular markers. While an aliquot of the reaction mix with the detection antibodies from the tube of the kit diffuses the nitrocellulose strip the capturing antibodies prepped to defined positions at the nitrocellulose bind also to the tumor marker, which is already bound by the detecting antibodies. Streptavidin and biotin make the signal visible or at least intensify the visibility of the signal at the defined position. The readout of the visible stripes on the predefined positions of the nitrocellulose track informs about the presence or absence of tumor markers in the sample tested and thus, allows a staging of captured cancer cells for prognosis.

[0044] According to a further embodiment the diagnostic kit of the invention is designed to use different or additional detecting antibodies. Instead of or additionally to the detecting antibodies for E cadherin, EPCAM and Vimentin other markers are used which target e.g. epithelial cells for detection of cancer, or e.g. mesenchymal cells for showing metastasis and / or cancer infiltration, or e.g. markers that will define the tissue specificity of cancer cells. For all these additional detecting antibodies there have to be also additional tracks on the nitrocellulose strip containing the corresponding capturing antibodies for said other markers. A readout of these augmented kits allows an even more precise diagnostic result and a more precise tumor staging. Not only can the origin of the tumor cells be characterized e.g. by suitable cellular markers for e.g. breast, urinary, bladder, prostate or colon cells, but also the stage of a tumor development can be characterized due to the read out of e.g. markers that indicate the metastatic potential.

[0045] This detailed readout of the diagnostic kit of the invention is particularly useful for the detection and / or prognosis of breast, cervix, urinary, bladder, prostate and / or colon cancer, or all cancers which release cancerous cells into body fluids, which can be extracted as liquid biological samples. As the whole process of analyzing the biological sample with the kit only takes

[0046] - for the first step of concentrating the tumor cells with the AK1223 nanoparticles between about 15 min to about 1 h,

[0047] - for the second step of detecting the tumor markers in the tube of the kit between another about 15 min to about 1 h, and

[0048] - for the capturing of the tumor markers on the nitrocellulose another 15 min to about 1 h, a final diagnostic result can be obtained in as less as 1 hour. However, even if the processing of the diagnostic kit is performed in a time frame of about 2 - 4 hours and a final result can be communicated to the patient in less than 1 day. Such a fast, simple and still highly specific diagnostic result is to most prominent advantage of the kit and method as described herein.

[0049] According to further and even more elaborate approach the diagnostic kit of the invention is particularly adapted to optimize a breast cancer diagnosis and does comprise a further or 2ndcassette for a parallel analysis of blood samples, which is suitable for determining and characterizing the molecular subtype of the breast cancer cells into Luminal A, Luminal B, Her2 positive and triple negative. The main advantages of the diagnostic kit as described herein and provided by the invention is the efficient and fast information on one or more of the following parameters: a) the presence of cancer, b) the presence of metastatic and / or invasive cell markers, c) the presence of receptors for estrogens (ER), progesterone (PR) and / or Her2. This detailed diagnosis allows for a personalized and fast adjustment or improvement of a potential therapeutic approach.

[0050] According to one working example the method for the detection and / or prognosis of cancer for solid tumors applies the following amounts and concentration of the substances or compositions as described above. The kit can be worked by

[0051] ® introducing the magnetic coated nanoparticle into 10mL to 40ml, preferably 20ml to 30ml, of liquid biopsy material for 10 min to 20 min at room temperature,

[0052] • isolating cancer cells bound to the nanoparticles and lysing them with a buffer by incubation for 5 min to 40 min, preferably for 10 min to 20 min,

[0053] ® introducing the resulting proteins into a tube containing lyophilized detecting monoclonal antibodies, e.g. for E cadherin, Vimentin and / or EPCAM, or any other alternative marker which target epithelial cells for detection of cancer, or mesenchymal cells for showing metastasis and / or cancer infiltration, or markers that will define the tissue specificity of the cancer, the secondary gold labelled monoclonal antibody, and gold labelled biotin;

[0054] • incubating the protein-antibody mix for 10 min to 1 h, preferably 20 min at room temperature,

[0055] • introducing the resulting mix to the cassette comprising the nitrocellulose strip coated with capturing monoclonal antibodies in different lanes, wherein the capturing antibodies recognize different epitopes than the detecting antibodies, and further comprising streptavidin,

[0056] • allowing the sample applied on the nitrocellulose strip to run by capillary action for 10 min at room temperature,

[0057] • visualizing the results as bands at the certain points where the different capturing antibodies have been applied.

[0058] The present invention thus, describes for the first time a novel nanoparticle that is a magnetic nanoparticle coated with AK1223, a Hypericum perforatum extract, which has the ability to adhere selectively on cancer cells, in a significantly greater extend compared to the non-cancerous cell lines (p<0.0005). The present invention also describes the use of the novel nanoparticle in a very accurate and sensitive diagnostic kit that can provide reproducible results from a liquid biopsy (urine or blood) within 1 hour, by use of the capacity of the afore mentioned novel nanoparticle coated with AK1223 as a cancer cell capturing tool, followed by lysis, using an appropriate lysis buffer (i.e. RIPA buffer), in combination with a strip containing certain capturing antibodies that are used for the detection and the staging of cancer.

[0059] Preferably, the cells captured by the novel nanoparticle of the present invention that is coated with AK1223, for use in the kit of the present invention are urinary bladder cells from urine, prostate, colon and breast cancer cells from blood. The present invention describes a magnetic nanoparticle that have been coated with a Hypericum perforatum extract (AK1223), for use in a diagnostic kit for liquid biopsies, where it isolates cancer cells.

[0060] Advantageously, the Hypericum perforatum extract (AK1223) for coating the novel magnetic nanoparticle is extracted from dry herb by soaking the plant material in solvent each time for 24 hours at room temperature, followed by degreasing in a separatory funnel so that the extract is washed to remove the greater percentage of fatty substance and the supernatant is then taken and evaporated to dryness at room temperature. Preferably, the solvent extraction is repeated one to three times. It is advantageous that for the creation of the novel magnetic nanoparticle of the present invention the Hypericum perforatum extract (AK1223) is extracted by use of Methanol or ethanol. Preferably, the Hypericum perforatum extract (AK1223) is evaporated to dryness at temperature ranging from 40°C to 60°C.

[0061] Advantageously is also an alternative aqueous extracted solution of Hypericum perforatum extract (AK1223) prepared by adding dry pulverized herb to boiling water and let it stay for 10 min; after the solution had cooled, the extract is infiltrated using filter paper and the extract is then followed lyophilization procedure to give lyophilized powder.

[0062] The present invention further describes a novel diagnostic kit, referred to also as “AK1223-kit”, comprising:

[0063] -the novel magnetic coated nanoparticle of the present invention as a cancer cell capturing tool,

[0064] -an appropriate lysing buffer (i.e. but not restricted to RIPA buffer), -a tube containing: lyophilized detecting monoclonal antibodies for E cadherin, Vimentin and EPCAM (all rabbit anti-human, at least 1 pg of each) or the other marker that may be used alternatively, as described in the present specification which may target epithelial cells (for detecting cancer) or mesenchymal cells (for showing metastasis and or cancer infiltration) or markers that will define the tissue specificity of the cancer and also the secondary gold labelled monoclonal anti-rabbit antibody (1 pg) and also gold labelled biotin; and

[0065] - a nitrocellulose strip containing the appropriate capturing antibodies used for the detection and the staging of cancer.

[0066] It is of advantage that in the diagnostic kit of the present invention, the nitrocellulose strips comprise at least three (3) different monoclonal antibodies which are necessary for the characterization of the isolated cancers and the streptavidin which will be used as an internal control. Preferably, the reproducible results of the diagnostic kit of the present invention are final within 1 to 2 hours.

[0067] Advantageously, the diagnostic kit of the present invention is used both for the detection and for the prognosis of cancer, but alternatively the kit may be used also for the detection of cancer alone and for the prognosis of cancer alone, in different applications and according to wish.

[0068] It is of particular advantage that the diagnostic kit of the present invention is used for use for the detection and / or prognosis of urinary bladder, prostate, colon cancer.

[0069] Advantageously, the diagnostic kit of the present invention, may also be used for the detection and / or prognosis of breast cancer, when this comprises an additional (2nd) cassette for analysis of blood samples in order to determine the molecular subtype (Luminal A, Luminal B, Her2 positive and triple negative) of breast cancer cells, which kit indicates a) the presence of cancer, and whether it is metastatic / invasive or not and b) the presence of receptors for estrogens (ER), progesterone (PR) and Her2, which will define the therapeutic approach in each case.

[0070] Preferably, in the diagnostic kit of the present invention lysis includes a step of removing red cells with erythrocyte lysis buffer (ELB) before reaching the step of the RIPA Buffer. Brief description of the Figures

[0071] The present invention is further explained in the following description for clarifying the correlation of the claimed examples as shown in the figures.

[0072] Figure 1 shows colon cancer cell staining with AK1223. In this case we used two different commercially available colon cancer cell lines the DLD-1 and the HT-129. AK122 contains a fluorescent compound that can be excited by light at a peak of 580 nm and it emits light at a peak of 620nm (Texas red). Figure 1 demonstrates the level of uptake of AK1223 by the two colon cancer cell lines. It was found that more than 95% of the cells in each case were stained by the AK1223. (DAPI: nuclear stain, Texas red: AK1223 stain).

[0073] Figure 2 shows breast cancer cell staining with AK1223. The breast cancer cell lines used were the MCF-7 and the MDA MB 231 . AK1223 contains a fluorescent compound that can be excited by light at a peak of 580 nm and it emits light at a peak of 620 nm (Texas red). Figure 2 demonstrates the level of AK1223 uptake by the two breast cancer cell lines. More than 95% of the breast cancer cells in each case were stained by the AK1223. (DAPI: nuclear stain, Texas red: AK1223 stain).

[0074] Figure 3 shows urinary bladder cancer cell staining with AK1223. The cell line used in this case was the T24. AK1223 contains a fluorescent compound that can be excited by light at a peak of 580 nm and it emits light at a peak of 620 nm (Texas red). Figure 3 demonstrates the AK1223 uptake in urinary bladder cancer cells. More than 95% of the cells were stained by the AK1223. (DAPI: nuclear stain, Texas red: AK1223 stain) The experiments mentioned in Figures 1 , 2 and 3 indicate the specificity of the AK1223 for breast, colon and urinary bladder cancer cells.

[0075] Figure 4 shows normal prostate and wild type Human Mesenchymal Stem Cells (HMSC) cell staining with AK1223. In this case both cell lines used were non-cancerous. The aim of this experiment was to determine the specificity of AK1223 for cancer cells. AK1223 contains a fluorescent compound that can be excited by light at a peak of 580 nm and it emits light at a peak of 620 nm (Texas red). Figure 4 demonstrates the level of AK1223 uptake by normal cancer cell lines. In both non -cancerous cell lines tested less than 10% of the cells were weakly stained by the AK1223. (DAPI: nuclear stain, Texas red: AK1223 stain). According to the experiment described in Figure 4 AK1223 is not uptaken by the non-cancerous cells. The conclusion obtained from the experiments described in Figures 1 , 2, 3, 4 indicates the sensitivity and the specificity of the AK1223 to recognize breast, colon and urinary bladder cancer cells. Based on these data, the AK1223 coated magnetic beads present a high specificity and sensitivity for cancer cells.

[0076] Figure 5: Based on the evidence from the experiments described in Figures 1 , 2, 3, 4, In order to determine if the AK1223 retains its sensitivity to cancer cells after the coating of magnetic beads, T4 human urinary bladder cancer cells were incubated with magnetic beads coated with the AK1223. The magnetic beads coated with the AK1223 entered the cancer cells in a similar manner to the AK1223 alone. More than 95% of the T4 bladder cells uptake the AK1223 coated magnetic beads.

[0077] Figure 6 shows HPLC results of a standard hypericin (98%) (6A) and HYP3 (6B)

[0078] Comparing the spectroscopic data with the existing literature on St. John's wort, we observe the existence of peaks corresponding to the main substances. Thus, in the spectrum with deuterated methanol solvent CD3OD, we observe peaks corresponding to hyperforin [Beerhues Ludger (2006) Hyperforin, Phytochemistry 67:2201 -2207] and are in a blue (square round edges) frame and peaks corresponding to flavonol (quercetin and kaemferol derivatives with sugar probably) [Tusevski O., Krstikj M., Stanoeva J.P, Stefova M., Gadzovska Simic S. 2018. Phenolic profile and biological activity of Hypericum perforatum L : Can roots be considered as a new source of natural compounds? South African Journal of Botany, 117, 301 -310] and are in a green (oval) frame. Finally, with a black (square) frame are the peaks [between 4.5 and 3.4] corresponding to sugars.

[0079] Examples and applications of the novel nanoparticle and diagnostic kit of the present invention are given in the present description indicatively, but not restrictively, for bladder cancer and for breast cancer. However, the novel nanoparticles that are coated with AK1223 and the diagnostic kit of the present invention are applicable to also other cancers which have the common feature of being solid tumors and which allow for the application of the same principles while testing liquid biopsy material. Examples

[0080] The following examples are provided solely for the purpose of illustrating the present invention and are not intended to limit its scope in any way with respect to the materials, methods, or conditions described.

[0081] Example 1 : Preparation of AK1223

[0082] Plant collection and identification:

[0083] The fresh aerial parts of Hypericum perforatum were collected from Western Macedonia in Voio, Greece. The voucher specimen has been identified at the Section of Pharmacognosy and Natural Product Chemistry in University of Athens, Greece.

[0084] Plant preparation:

[0085] The lower parts of the collect samples were discarded and the flowering tops with small parts of the dry shoots were chosen and further pulverized.

[0086] Preparation of two different suitable extracts:

[0087] Methanolic / ethanolic extract of the herb was prepared by extracting 10 g of dry herb with 100 mL methanol / ethanol. Solvent extraction was repeated three times by soaking the dry plant in 100rriL methanol each time at room temperature for 24 hours (AK1223-1 ). Degreasing in a separatory funnel was followed so the methanolic extract was washed three times with petroleum ether (100rriL each time) to remove the high percentage of fatty substances. The formatting upper layer is the ethereal layer and is green. The methanolic layer (red color) creates a characteristic emulsion (gray color) which makes the process difficult, so it is left alone and the supernatant is taken. The procedure was repeated three times (AK1223-2). The obtained extracts were evaporated to dryness, using a Rotavapor Buchi R-200 vacuum condenser in a Buchi Heating Bath B-490 at temperatures ranged from 40°C to 60°C, resulting in 1 ,5 g of dry extract (AK1223-3). The extraction yield of this procedure is 15%, in comparison to other published method that shows only 9,5% (EP 1522309), representing 58% more efficiency compared to other published.

[0088] Aqueous extracted solution was prepared by adding 2 g of dry pulverized herb to 300 mL of boiling water and let it stay for 10 min. After the solution had cooled, the extract was infiltrated using filter paper and the sample is then followed lyophilization procedure to give 510 mg of lyophilized powder. This method is the most efficient since it gives an extraction yield of 25% (AK-1223-4).

[0089] For the methanolic / ethanolic extract were needed 3 days but the present invention reached an increased production, which is requested in the case of industrial applicability of the invention. It is also advantageous that the present invention can use room temperature. This has the advantage that energy and appliances are thus spared.

[0090] It is also advantageous although not necessary that at the end the AK1223 extract of the present invention is to be dried, for reasons of better handling such, for example one can create any concentration needed, use any solvent , preferably but not necessarily methanol. Alternative solvents would be ethanol, water.]

[0091] Example 2: Coating of the nanoparticles

[0092] For this, magnetic nanoparticles are placed in a 100 mL round-bottom flask and 10 mL of Sera-Mag (Magnetic particles , Sigma-Aldrich) (50 mg / mL) are added, where they are left under stirring for about 10 minutes.

[0093] Then, 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is added and the solution is stirred for 30 minutes. Finally, DMAP (4-Dimethylaminopyridine) and AK1223 powder dissolved in DMSO (dimethyl sulfoxide) are added. For this, a 0,2-0,5gr AK1223 were used.

[0094] The reaction solution remains under stirring for 2 days. Purification is carried out using the dialysis method on a 3500 kDa membrane in order to remove any amount of unreacted AK1223 and DMSO. A total of 5 changes are made every three hours in the external Mi II iQ water of about 2 L and is completed in 3 days.

Claims

Claims:1 . A method to produce AK1223 coated magnetic nanoparticles, comprising the steps of:- Producing an herbal preparation by soaking dry Hypericum perforatum herbs for at least 10 minutes to 24 hours at room temperature in a solvent preferably hot water and / or alcohol; degreasing the alcoholic herbal preparation in a separatory funnel to remove fatty substance; allowing the herbs to settle or centrifuging the herbal preparation; extracting the supernatant and evaporating remaining solvent to receive an AK1223 concentrate or powder;- Incubating the magnetic nanoparticles with an AK1223 extract prepared from the AK1223 concentrate or powder; and- Drying the coated magnetic nanoparticles.

2. A magnetic nanoparticle coated with a degreased Hypericum perforatum extract (AK1223) for use in a method to isolate or concentrate cancer cells from liquid biological samples or for use in a diagnostic kit, wherein the Hypericum perforatum extract (AK1223) is extracted from dry herb by soaking the plant material in solvent each time for 24 hours at room temperature, followed by degreasing in a separatory funnel so that the extract is washed to remove the greater percentage of fatty substance and the supernatant is then taken and evaporated to dryness at room temperature.

3. The magnetic nanoparticle according to claim 1 wherein solvent extraction is repeated one to three times.

4. The magnetic nanoparticle according to claims 1 wherein an aqueous extracted solution of Hypericum perforatum (AK1223) is prepared by adding dry pulverized herb to boiling water and let it stay for 10 min, after the solution had cooled, theextract is infiltrated using filter paper and the extract is then followed lyophilization procedure to give lyophilized powder.

5. A diagnostic kit for capturing cancer cells from liquid biological samples comprising:- the magnetic nanoparticle coated with the degreased or aqueous solution of Hypericum perforatum extract (AK1223) as a cancer cell capturing tool,- an appropriate cell lysis buffer;- a tube containing a.) one or several lyophilized monoclonal antibodies for the detection of tumor markers hereinafter identified as detecting antibody, and optionally the detecting antibody is a rodent anti-human antibody, b.) a secondary gold labelled monoclonal anti-rodent antibody, and c.) gold labelled biotin;- a nitrocellulose strip containing in different lines a.) one or several alternative capturing antibodies used for the detection of specific proteins of the cells previously isolated with the AK1223 coated magnetic nanoparticles, wherein the capturing antibody binds to a different epitope of the aforementioned proteins (tumor markers) [and is optionally also bound by the detecting antibody], and b.) streptavidin.

6. The diagnostic kit according to claim 5 comprising:- the magnetic nanoparticle coated with the degreased or aqueous solution of Hypericum perforatum extract (AK1223) as a cancer cell capturing tool,- an appropriate lysis buffer (i.e. RIPA);- a tube containing a.) lyophilized detecting monoclonal antibodies for E cadherin, Vimentin and / or EPCAM, UPKII wherein the detecting antibodies are rabbit antihuman in a concentration of at least 1 pg of each, b.) a secondary gold labelled monoclonal anti-rabbit antibody in a concentration of about 1 pg andc.) gold labelled biotin; a nitrocellulose strip containing the one or several capturing antibodies used for the detection of the cells captured by the detecting antibody and / or [cl10] for the staging of captured cancer cells for prognosis.

7. The diagnostic kit according to claim 5, wherein instead of or additionally to the detecting antibodies for E cadherin, EPCAM and Vimentin other markers are used which target epithelial cells for detection of cancer, or mesenchymal cells for showing metastasis and / or cancer infiltration, or markers that will define the tissue specificity of cancer cells, and where also the nitrocellulose strip contains the corresponding capturing antibodies for said other markers8. The diagnostic kit according to claims 4 to 6 for use for the detection and / or prognosis of urinary bladder, prostate and / or colon cancer.

9. The diagnostic kit according to claims 4 to 6, for use for the detection and / or prognosis of breast cancer.

10. The diagnostic kit according to claim 8 characterized in that an additional, 2ndcassette for analysis of blood samples is included into the kit, which is suitable for determining and characterizing the molecular subtype of the breast cancer cells into Luminal A, Luminal B, Her2 positive and triple negative.

11. The diagnostic kit according to claim 8 or 9, wherein the kit indicates a) the presence of cancer, and the presence of metastatic and / or invasive cell markers b) the presence of receptors for estrogens (ER), progesterone (PR) and Her2, and thereby improve a potential therapeutic approach.

12. The diagnostic kit according to claims 4 to 10, where lysis includes a step of removing red cells with erythrocyte lysis buffer (ELB) before reaching the step of the RIPA Buffer.

13. A method of capturing and characterizing cancer cells from a liquid biological sample for the detection and / or prognosis of cancer wherein the circulating tumor cells (CTCs) are isolated by use of the magnetic nanoparticles according to claims 1 to 3, then lysed with a cell lysis buffer, e.g. RIPA buffer and characterized via antibody detection of tumor markers.

14. The method for the detection and / or prognosis of cancer according to claim 12 for solid tumors by use of the kit of claims 4 to 11 , characterized in that it comprises the following steps:- the magnetic coated nanoparticle are introduced into 10mL to 40ml, preferably 20ml to 30ml, of liquid biopsy material for 10 min to 20 min at room temperature,- cancer cells bound to the nanoparticles are isolated and lysed with buffer by incubation for 5 min to 40 min, preferably for 10 min to 20 min,- the resulting proteins are then introduced into a tube containing a.) lyophilized detecting monoclonal antibodies, e.g. for E cadherin,Vimentin and / or EPCAM, or any other alternative marker which target epithelial cells for detection of cancer, or mesenchymal cells for showing metastasis and / or cancer infiltration, or markers that will define the tissue specificity of the cancer, b.) the secondary gold labelled monoclonal antibody, and c.) gold labelled biotin;- the protein-antibody mix is incubated for 10 min to 1 hr, preferably 20 min, at room temperature,- the resulting mix is then introduced to the cassette comprising a.) a nitrocellulose strip coated with capturing monoclonal antibodies in different lanes, wherein the capturing antibodies recognize different epitopes than the detecting antibodies, and b.) streptavidin as a functional control, so that they are able to capture E cadherin, Vimentin and / or EPCAM in the case of cancer detection,- the sample applied on the nitrocellulose strip is allowed to run by capillary action for 10 min at room temperature,- the results are visualized as bands at the certain points where the different capturing antibodies have been applied.

15. The method according to claims 12 and 13 for use for the detection and / or prognosis of breast cancer, characterized in that there is an additional step of using an additional (2nd) cassette for analysis of blood samples in order to determine the molecular subtype of breast cancer cells as Luminal A, Luminal B, Her2 positive and triple negative, and which method also indicates a) the presence of cancer, and whether it is metastatic and / or invasive, b) the presence of receptors for estrogens (ER), progesterone (PR) and Her2, which will allow a personalized therapeutic approach in each case.

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