Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Photoactivatable nanoparticles for photodynamic applications method of preparation thereof, pharmaceutical compositions containing them, and use thereof
• the present invention relates to photoactivatable nanoparticles for photodynamic applications, method of preparation thereof, pharmaceutical compositions containing them, and the use thereof as medicaments in photodynamic therapy.
• Photodynamic therapy is a relatively new method of treating various diseases, with a particular focus on the treatment of tumors, for example squamous cell carcinoma of the head and neck, PDT consists in combination of photosensitizer (PS), molecule of oxygen, and subsequent laser irradiation of tumor lesions. After laser irradiation of the tumor, the molecules of the photosensitizer absorb the photons of light and subsequently excite oxygen while releasing singlet oxygen, which is highly destructive causing damages of the cellular structures, such as DNA or membranes in close surrounding.
• Efficiency of PDT largely depends on the photochemical, photobiological, and phototherapeutic properties of PS.
• Critical factor is the formulation of PS, which ensures, that the effect is selectively focused on the target tumorous tissue.
• PDT is a preferred method of treatment not only in tumorous, but also in many non-tumorous applications, for example in cosmetics, where the photosensitizer is combined with a biological filling material (comprising collagen, hyaluronic acid, and other synthetic or natural products) and the fonnulation is subsequently injected to the treated location (US201 1002 973 A 1).
• PDT is used also as a method of treatment of unwanted cellulitis (US8414880 B2) or in the prevention and treatment of vascular or inflammatory diseases (US20090306032 Al), where the authors describe solid irpidic nanoparticles obtained by micro-emulgation in heat, containing cholesteryl propionate and/or cholesteryl butyrate.
• Nanomedicine is a fast developing discipline of medical research, which is focused especially on the development of nanoparticles (NPs) for prophylactic, diagnostic, and therapeutic applications.
• the size of nanoparticles is generally usually defined in the range of 1 nm tiara 1000 nm.
• Nanomedicine functions on the same principle as many biological processes and cellular mechanisms. Photosensitizers incorporated inside the nanoparticles provide a very promising approach in solving the problem of both bioavailability and targeted deliver ⁇ - to the tumorous tissue. Methods and procedures for the synthesis, functionalization and the use of NPs are today widely studied and they represent new strategies for molecular targeting, individual therapy and minimally invasive diagnostic techniques.
• PSs are hydrophobic or amphiphilic substances usually insoluble in water.
• PSs are hydrophobic or amphiphilic substances usually insoluble in water.
• PSs are administered in alcoholic solution (ethanol, propylene glycol).
• alcoholic solution ethanol, propylene glycol.
• Temoporfin Bioiitec Pharma, Scotland, United Kingdom. was approved in EU for palliative tumors of head and neck, tumors prostate and pancreas (US20060040912 Al).
• Solid lipid nanoparticles with stabilized surface with non-covIERly bound medicaments represent a new type of medical formulation.
• these SLNPs after adhesion to apolipoproteins, imitate low-den sity-lipoprotein (LDL) transporting the lipids to the tissues.
• LDL receptors are overexpressed on the surface of many tumorous cells (Kretzer, lara F., Durvanei A. Maria, and Raul C. Maranhao. 2012.
• SLNPs are then an effective system of administration of medicaments improving biological availability also for the substances poorly soluble in water.
• Formulations on the basis of SLNP provide generally higher bioavailability and a higher therapeutic effect in vitro and in vivo in comparison with commercial formulations, especially for use in intravenous drug application form.
• Lipid particles that have the diameter smaller than one micrometer. Lipid particles were obtained by adding heated lipids (above their melting point) to the mixture composed of water, surfactant, and co-surfactant.
• Patent application WO 1993005768 Al describes a system of drug delivery based on solid particles of lipidic materials or mixtures thereof, with the diameter from 10 ran to 10 ⁇ . They provide longer time of controlled release of acti ve substance and enable incorporation of hydrophilic drugs to the solid core of the particle.
• US20080090803 Al describes covalent conjugates of fatty alcohols and pharmaceutical carriers for the treatment of tumorous and viral diseases and psychiatric disorders.
• US20060127471 Al describes the preparation of liposomal formulations containing hydrophobic photosensitizer (temoporfin) and their use in therapy, particularly using intravenous application.
• Phospholipids are modified by PEGylation [containing poly(ethylene oxide) as an integral part .
• the resulting PEGylated liposomes include a hydrophobic photosensitizer within the membrane. This mechanism delivers the photosensitizer to the cellular membrane of the system directly to the site of action.
• US7354599 B2 describes pharmaceutical liposomal formulations for PDT that contain a hydrophobic photosensitizer of porphyrin type (temoporfin), monosaccharide (fructose and glucose) and one or more synthetic phospholipids. Thanks to the lyophilization process, the resulting formulations are stable on storage and suitable for intravenous administration.
• US8709449 B2 describes the formulation increasing the efficiency of PDT and reducing phototoxic reactions of skin caused by PDT.
• the lipid emulsion contains soybean oil, triglycerides with medium long chains and phospholipids from eggs as hydrophobic components.
• PS is selected from die group of porphyrins, phthalocyanines, metallo-graphite derivatives, dyes and synthetic photosensitize rs.
• US20080311214 Al describes modified lipidic nano- /micro-particles that contain on their surface a molecule or a ligand targeting to a specific binding site, and their use for peroral administration of medicaments and antigens.
• Patent application WO 2006069985 A2 describes methods of preparation of lipid-based nanoparticles by homogenization for the purpose of incorporating a compound into the already pre- prepared nanoparticles using a dual asymmetric centrifuge.
• Patent application WO 2010112749A1 describes the method of preparation and composition of suspensions of solid lipidic nanoparticles in aqueous phase, containing the minoxidil in a lipid matrix (mono-, di- and triglycerides non-containing stearic acid).
• SLNPs contain at least one amphiphilic compound selected from phospholipids (soy lecithin, composed mainly of phosphatidylcholines).
• SLNPs contain mainly phosphatidylcholine, lecithin and one or more surfactants (selected from esters of fatty acids with sorbitans) and water phase (comprising water, propylene glycol and eventually ethanol).
• the authors focus on the cosmetic and pharmaceutical use.
• Patent application WO 201 1 116963 A2 describes a system of delivery' of medicaments using SLNP that - as lipidic carriers - comprise at least one active substance and are coated with polymers.
• the active substance is selected from cosmetic, pharmaceutical and/or alimentary active substances and/or adjuvans.
• Polymers (prepared by simple or complex coacervation) are selected from the group of proteins, polysaccharides, polyesters, polyacrylates, polycyanoacr lates, and/or mixtures thereof.
• US5785976 A describes the preparation of colloidal suspensions of solid lipidic particles of anisometric shapes with a lipid matrix which is in a stable polymorphic modification. They further comprise a suspension of micro- and submicron particles of bioactive compounds. These suspensions were prepared especially for parenteral administration, which is very convenient for bioactive substances poorly soluble in water, especially medicaments, and their use in cosmetic, food and agricultural industries.
• US4880634 A describes lipidic nano-pellets containing pharmacologically active substances, the size of which is in the range of from 50 to 1000 nm, and may be used as adjuvants in the system of transport of drugs for peroral administration.
• PCT/EP2012/055222 (WO 2012/127037) describes a pharmaceutical composition, which comprises a corticosteroid and/or vitamin D derivative incorporated as a solid solution or dispersion in lipid nanoparticles, said lipid nanoparticles being solid at ambient temperature and comprising a first lipid with a melting point above body temperature, and a pharmaceutically acceptable surfactant.
• US 2015/0273084 Al relates to a methylene blue nanoparticie for bioimagmg and photodynamic therapy, and a use thereof as a cancer therapeutic agent and a contrast agent.
• the nanoparticie contains a methylene blue - fatty acid (or salt thereof) complex and an aphiphilic polymer (polyoxyethylene-polyoxypropylene block copolymer), wherein the methylene blue - fatty acid complex is enclosed in an amphiphilic polymer micelle (Pluronic F-68).
• Xingwang Zhang, Huan Wang, Tianpeng Zhang, Xiaotong Zhou, Baojian Wu, European Journal of Pharmaceutical Sciences 62 (2014) 301-308 describes the preparation, solubilization, stabilization and bioavailability of polymeric mixed micelles with stiripentol as model drug.
• the micelles were prepared by rapid dispersion of an ethanol solution containing stiripentol, monomethoxy poly(ethylene glycol)-b-poly(8-caprolactone) and sodium oieate into water.
• nanoparticles there may be fundamental also other characteristics, for example the method of administration of a medicament, colloidal stability and interaction with blood proteins, biodegradability of the carrier, low incorporation and adsorption of die curative substance, complex development of methodologies for the control of quality of the transporting system, problems with stability during storage, toxicological tests of some types of nanoparticles are not fully completed and there is often need to increase the efficiency of the production (W. Mehnert and K. Mader, Advanced Drug Delivery- Reviews, Vol. 47, No. 2-3, pp. 165-196, 2001 ; E. B. Souto, P. Severino, M. H. A. Santana, and S. C. Pinho, Quimica Nova, Vol. 34, No. 10, pp. 1762- 1769, 2011).
• the present invention provides fully biodegradable formulation of photoactivatable nanoparticles for photodynamic applications with minimum side effects. It is based on solid lipid nanoparticles (SLNPs) of the photosensitizer and fatty alcohols, whereas the surface of these solid lipid nanoparticles is stabilized with a non-toxic surface active polymer, such as a copolymer po3y(ethyiene oxide) monom ethyl ether - block - poly(8-caprolactone), which prevents entrapping of SNLPs to reticuloendothelial system .
• SLNPs solid lipid nanoparticles
• a non-toxic surface active polymer such as a copolymer po3y(ethyiene oxide) monom ethyl ether - block - poly(8-caprolactone
• Fatty alcohols are then fully degradable during metabolization by ⁇ -oxidation, while the surface active copolymer itself is also biodegradable to the 6-hydroxyhexanoic acid and poly(ethyiene oxide), and based on the relatively low molecule weight, both types of fragments are eliminable by kidneys, SLNPs are at the storage temperature of 4 °C solid, stable, and do not aggregate. Melting temperature of the core is 39 °C, which causes releasing of the photosensitize!- in the tumor in a controllable way as a result of intensive hyper thermic metabolism.
• the subject of the present invention is a photoactivatable nanoparticle for photodynamic applications, containing a core and a shell, the core of which contains from 1 to 50 weight % of a photosensitizer, referred to the total dry weight of the nanoparticle, preferably from 1 to 4 weight % of the photosensitizer, referred to the total dry weight of the nanoparticle, and the said photoactivatable nanoparticle further contains from 1 to 80 weight % of (Cg to C 22 ) fatty alcohols, referred to the total dry weight of the nanoparticle, preferably from 12 to 72 weight % of (Cs to C 22 ) fatty alcohols, referred to the total dry weight of the nanoparticle; and the shall contains from 1 to 80 weight % of polymeric surfactant, preferably of polyethylene oxide) monomethyl ether - block - poly ( ⁇ -caprolactone), referred to the total dry weight of the nanoparticle, preferably the shell contains from 14 to 72 weight % of the said polymeric surfactant,
• the core of the nanoparticle is solid at the temperature of 4 °C, and liquid at the temperature of 39 °C, determined by differential scanning calorimetry with flushing with nitrogen gas with the volume of 50 cm7min while using indium as a standard in temperature cycles: heating - cooling - heating, at 5 °C/min, and the temperature reading was performed from the first curve of heating of the differential scanning calorimetry, where the result was a bimodal thermogram with two endotherms, one for the eutectic mixture of the photosensitizer and the fatty alcohol, and the second one for the fatty alcohol .
• Inclusion of temoporfin to the lipidic matrix leads to the melting temperature around 26 °C, when the eutectic mixture of the photosensitizer and of the (Cs to C 22 ) fatty alcohol melts, and between 35 and 39 °C, preferably between 37 and 39 °C, most preferably at around 39 °C, when the fatty alcohol-based core matnx itself melts. It means, that two phases exist in the lipid core, containing the photosensitizer and the (Cg to C 22 ) fatty alcohol. ' This composition enables the stability of solid lipid nanoparticies during their storage at the temperature of 4 °C, and, at the same time, the melting tem perature of the core of 39 °C.
• the photosensitizer is selected from derivatives of chlorine and/or porphyrin and/or anthraqumone, more preferably the photosensitizer is selected from the group comprising temoporfin, verteporfin, lutetium texaphyrin, hypericin and porphycene.
• the core of the photoactivatable nanoparticle contains one type of (CH to C22) fatty alcohol; preferably the fatty alcohol is the (CM) fatty alcohol.
• the core of the photoactivatable nanoparticle contains a combination of (Cs to C22) fatty alcohols.
• the polymeric surfactant is the poly(ethylene oxide) monomethy] ether-Moefc-polyie-caprolactone), preferably with the degree of polymerization of the poly(ethylene oxide) block in the range of from 10 to 100, and with the degree of polymerization of the poiy(e-caproiactone) block in the range of from 3 to 60, more preferably the degree of polymerization of the poly(ethylene oxide) block is in the range of from 20 to 50, and the degree of polymerization of the poly(e-caprolactone) block is in the range of from 3 to 20; most preferably the degree of polymerization of the poly(ethylene oxide) block is 45, and the degree of polymerization of the poly(s-caprolactone) block is 7.
• Tire subject of the present invention is also a method of preparation of the photoactivatable nanoparticle according to the present invention, wherein:
• the photosensitizer is dissolved in a solvent, preferably in dichloromethane, and it is added to the (Cg to C22) fatty alcohol heated to 45 °C while forming a first phase;
• the polymeric surfactant is dissolved in an organic solvent, preferably in dichloromethane, then water is added, and the mixture is heated to 45 °C while forming a second phase;
• both heated phases are mixed together and homogenized, preferably for the period of 2 minutes at 8000 rotations per minute (RPM);
• the resulting emulsion is cooled down to the laboratory temperature.
• Ultrasound sonication may be performed, for example, at the laboratory temperature and the ultrasound amplitude between 50 % and 75 %, preferably at the ultrasound amplitude of 50 % for the time period of 35 mm and then at the ultrasound amplitude of 70 % for the time period of 5 min.
• the subject of the present invention is also a pharmaceutical composition which comprises the photoactivatable nanoparticle according to the present invention, and the said pharmaceutical composition further comprises pharmaceutically acceptable additives, selected from the group comprising anti-adhesive agents, binders, coating agents, coloring agents, swelling agents, flavoring agents, lubricants, preservatives, sweeteners, cryoprotectives, absorbents.
• pharmaceutically acceptable additives selected from the group comprising anti-adhesive agents, binders, coating agents, coloring agents, swelling agents, flavoring agents, lubricants, preservatives, sweeteners, cryoprotectives, absorbents.
• the subject of the present invention is also the photoactivatable nanoparticle according to the present invention and/or the pharmaceutical composition according to the present invention for the use as a medicament.
• the subject of the present invention is also the photoactivatable nanoparticle according to the present invention and/or the phanriaceutical composition according to the present invention for the use as a medicament for intravenous applications.
• An intravenous application is understood as injection of a liquid form of the medicament directly into a vein or a artery of a patient.
• the subject of the present invention is also the photoactivatable nanoparticle according to the present invention and/or the pharmaceutical composition according to the present invention for the use in the photodynamic therapy and/or photodynamic diagnostics.
• the present invention of the system of solid lipid nanoparticles provides a solution to overcome all of the aforementioned limitations of the background art, such as for example simple preparation, stability during the period of storage, the core of the nanoparticle at the storage temperature (4 °C) is solid, stable, and it does not aggregate at the physiologic pH (pH ⁇ 7,4), while the core melting temperature of 39 °C allows for release of the photosensitizer in the tumor in a controlled manner as a result of intensive hyperthermic metabolism.
• the nanosystem of this unique composition is fully biodegradable and it provides an increase of bioavailability and of the therapeutic effect in vitro and in vivo (in comparison with commercial formulations).
• the use of the nanoformulation is suitable both for the peroral as well for the intravenous medicament application forms.
• Figure 2 Thermograms of the DSC analysis of the solutions without a content of temoporfin (marked as SLNP with the corresponding ⁇ ), with incorporated temoporfin (prepared in accordance with the Examples 1, 2, and 3), and the pure temoporfin ( 10 to 20 mg).
• Figure 3 Transmission electron micrographs of samples of solid lipid nanoparticles with incorporated temoporfin, prepared in accordance with the Examples 1, 2, and 3.
• Figure 5 The influence of "one-shot" PDT on the growth of a human breast carcinoma Sine MDA- MB-231 .
• the tumorous areas were irradiated ( 100 J/cm 2 ) and subsequently dimensions of the tumors were measured regularly.
• Figure 6 The tumor growth inhibition curve - TGI % as a function of time after intravenous application (i.v.) of tested formulations of solid lipid nanoparticles with incorporated temoporfin prepared in accordance with the Examples 1 , 2, and 3 (the dose of temoporfm 0.8 mg/kg mouse) in comparison with the commercial formulation (Originator, containing 4 mg of temoporfm, 376 mg of ethanol, and 560 mg of propylene glycol per gram).
• the synthesis of the copolymer poly(ethylene oxide)-fc/oc£-poly(e -caprolactone) (PEO-&-PCL) is based on the ring-opening polymerization of ⁇ -caprolactone without the presence of a catalyst, initiated using the poly(ethylene oxide) monomethyl ether (MPEO), weight-average molecular weight 2 kDa.
• MPEO poly(ethylene oxide) monomethyl ether
• DP(PCL) is a degree of polymerization of the poly(e-caprolactone) block
• DP(PEO) is a degree of polymerization of the poly(ethylene oxide) block
• c and a are integral intensities of signals of the corresponding protons (see Figure 1).
• the length of the PCL block with the degree of polymerization DP 7 monomeric units.
• the resulting copolymer (polymeric surfactant) has therefore average formula: PEO 45 PCL 7 .
• Solid lipid nanoparticles with incorporated photosensitizer temoporfin were prepared using a high- power homogenization and ultrasound method.
• Lipid phase composed of 1-tetradecanol ( 10 mg/ml) was put in a glass vial and wanned to 45 °C.
• Temoporfin 1 mg/ml was dissolved in 1 ml of dichloromethane and added to the lipid phase to obtain the iipid-active pharmaceutical ingredient mixture after evaporation of the dichloromethane.
• Water phase was obtained by dissolving PEO 45 PCL 7 copolymer (50 mg/ml) in 1 ml of dichloromethane, subsequent addition of 5 ml of H 2 0 and warming up to 45 °C.
• the hot lipid phase was added to the hot water phase and subsequently the mixture was homogenized during the period of 2 min at 8000 RPM.
• the resulting emulsion was subjected to ultrasound using the standard ultrasound tip (T25 digital Ultra-Turrax, IKA, Schoeller Instruments, s.r.o., Czech Republic).
• the amplitude of the ultrasound was set to 50 % for the period of time of 35 min and to 70 % for the period of 5 min.
• the resulting emulsion was immediately cooled to room temperature.
• Nanoform illations were placed to T-zero hermetic aluminum pans and the analysis was done in temperature cycles: heating - cooling - heating, from 10 °C to 50 °C, at 5 °C/min. Melting temperatures of these samples were read from the 1st DSC-curve of heating. Inclusion of temoporfin into the lipid matrix results in bimodai thermogram with two endotherms: one at around 26 °C (eutectic mixture of temoporfin and 1 -tetradecanol) and another at around 39 °C i ltetradecanol). It means that there exist two phases in the lipid core, containing temoporfin and 1- tetradecanol.
• the composition according to the present invention enables the stability of solid lipid nanoparticies at storage temperature of 4 °C, and, equally, the core melting temperature at 39 °C.
• the tested nanoformulations were stored in glass vials in the refrigerator at temperature of 4 to 8 °C for the period of 1 month.
• DLS was used to measure the size of particles (Rj-i, nm), PDI, and ZP (mV), at pH ⁇ 5 and 7.4.
• a 0.05 was used as a threshold value of statistical significance.
• the tumor cell lines 4T1 (murine mammary carcinoma) and MDA-MB-231 (human breast adenocarcinoma) were maintained in the exponential phase of growth under sterile conditions in the medium RPMI 1640 enriched with 10 % of fetal calf serum (FCS), penicillin ( 100 units/ml), streptomycin (0,1 mg/ml), and L-glutamine (2 mmol). They were incubated at 37 °C in the atmosphere of 95 % air and 5 % CO2. Cell lines were passaged every second or third day.
• the cells 4T1 (2 x 10 s ) were placed in Petri dishes (35 mm) and incubated overnight at 37 °C. Then the tested formulations were added to the cells so that the final concentrations of temoporfin were within the range of from 0.25 to 2 uM, and subsequently they were further incubated for 16 h.
• the concentration of temoporfin in SLNP was chosen in order not to cause more than 10 % cell mortality on its own.
• Testing dishes were flushed with medium without phenol red (PR), and after addition of cultivation medium without PR they were irradiated for 30 min with halogen lamp (75 W) equipped with the filter for wavelengths of 620 to 680 nm (Andover, Salem, NH).
• the intensity of irradiation of ceils was 3.7 mW/cm 2 with the overall irradiation dose of 6.6 J/cm 2 .
• the ratio of live and dead cells was determined in Biirker chamber 24 h after irradiation using the absorption of trypan blue, thus obtaining the dose - effect curve, from which the values of IC 50 were numerically evaluated.
• the value of IC 50 represents the concentration of temoporfin, which is necessary to kill 50 % of cells, and it was calculated for each curve (Table 3).
• their toxic effect without irradiation was evaluated (the so-called "dark toxicity", toxicity without the photodynamic effect) and also the toxicity of SLNP formulations without the content of temoporfin ("empty", i.e. 1-tetradecanol and PEOxPCLy) at the same concentration conditions.
• a very low toxicity was found without irradiation as well as in case of SPLP without temoporfin (below 0 % in the entire concentration range).
• Subcellular localization of the photosensitizer of temoporfin was studied using fluorescence microscopy in comparison with the formulation of the originator (commercial, containing 4 mg of temoporfin, 376 mg of ethanol, and 560 mg of propylene glycol per gram).
• Tumor cells 4T1 inoculated to cover glasses, placed in Petri dishes (diameter 35 mm) were incubated with the formulations of temoporfin with the final concentration of 2 ⁇ (1 h or 4 h incubation) or 1 LLM (16 h of incubation) in the cultivation medium at 37 °C.
• the dishes with the cells were washed three times with PBS, poured with colorless medium without PR, and investigated under the microscope (DM IRB, Leica) equipped with the camera (DFC 480) while using the object-lens with 63-times oil immersion and filtration cube (N2.1, Leica), excitation: BP 515-560 nm, emission: LP 590 nm). Fluorescence of the formulations of the photosensitizer of temoporfin in the tumor 4 ⁇ cells was measured 1, 4, and 16 h after the addition of the formulation into the cultivation medium . The tested nanoformulation, in comparison with the formulation of the originator, has shown significantly faster kinetics of fluorescence growth (after 4 ).
• the ceils were incubated with tested nanoformulation of temoporfin, and 100 nM of MitoTracker Green (to label mitochondria) or 250 nM of ER-Tracker Blue to label endoplasmic reticulum (ER) was gradually added to the cells for 30 minutes. In order to label lysosomes, 500 nM of LysoTracker Green were added to the cultivation medium (1 h).
• Filtration cube Type A (Leica, excitation: BP 340-380 nm and emission: LP 400 nm) was used for the analysis of ER Tracker Blue; filtration cube 13 (Leica, excitation: BP 450-490 and emission: LP 515) was used for MitoTracker Green and LysoTracker Green. The tested nanormulation was localized mainly in ER.
• the area of the tumor (2 cm 2 ) was irradiated using a xenon lamp, (500-700 nm, ⁇ ⁇ . 652 nm), with the total maximum dose of light 100 J/'cm 2 and the intensity of irradiation of 200 mW/cm 2 .
• the control group of mice was irradiated under the same conditions but without the action of the medicament (see Figure 5).
• Each experimental group consisted of 5 mice. Forty-two days after administration of the tested nanofonnulations, the mice were sacrificed by overdose of ketamine and the tumors were evaluated histologically.
• the tumor growth inhibition (% TGI) as a measure of efficiency of the therapy was compared with the commercial fonnulation (containing 4 rag of temoporfin, 376 rag ethanol, and 560 rag of propylene glycol per gram), (refer to Figure 6). It was calculated in accordance with the equation (5), wherein V is the mean volume of the tumor of the specific group under test at the given time, Vo is the mean volume of the tumor of the specific group under test at the beginning of the experiment, V 3 ⁇ 4 is the mean volume of the tumor of the control group (with the application of physiological solution) at the given time, and V ko is the mean volume of the tumor of the control group at the beginning of the experiment:
• the fonnulation was analogical to Example 1 , the modification consisted in other composition (degree of polymerization of the poly ( ⁇ -caprolactone) block) of the copolymer used (PEO45PCL17).
• the results from the DLS analysis (R 3 ⁇ 4 PDI, ZP) are shown in Table 1.
• the results from DSC are shown in Figure 2.
• the results for the effectivity of encapsulation of temoporfin (%) and the contents of temoporfin (%) in SLNP are shown in Table 2. Transmission electron micrographs of the tested samples are shown in Figure 3.
• the time profile of in vitro releasing of temoporfin (%) from the nanoformulations is shown in Figure 4.
• Example 2 The analysis of the in vitro cytotoxicity was done in accordance with Example 1, the modification consisted in the use of the final concentrations of temoporfin incorporated in the SLNPs under test, which were in the range of from 1 to 20 uM. The results from the mortality of the cells (%), TC 50 ( ⁇ ) are listed in Table 3.
• the procedure of subcellular localization of the photosensitizer of temoporfin using the fluorescence microscopy was performed in accordance with Example 1.
• the procedure of determination of the subcellular co- localization of the nanoformulations using fluorescence microscopy was performed in accordance with Example I, the modification consisted in its localization - mainly in lysosomes. Tire results of therapeutic efficiency for the targeted transport of temoporfin with the application of PDT in vivo are shown in Figure 5 and their statistical evaluation is shown in Table 4.
• the graph showing the tumor growth inhibition is shown in Figure 6, Example 3
• Formulation in accordance with Example 1 its modification lies in weight composition of the lipidic phase, wherein the weight of 1 -tetradecanol was 50 mg/ml, and in the weight composition of the aqueous phase, where the weight of PEO 45 PCL 7 was 10 mg/ml.
• the results from the DLS analysis (3 ⁇ 4, PDI, ZP) are shown in Table 1.
• the results for the effectivity of encapsulation of temoporfin (%) and the contents of temoporfin (%) in SLNP are shown in Table 2.
• Modification lies in weight composition of the lipid phase, where the weight of 1 -tetradecanol was 50 mg/ml, and in the weight composition of the aqueous phase, where the weight of PEO45PCL17 was 10 mg/ml.
• the modification further lies in other composition (degree of polymerization of the poly ( ⁇ -caprolactone) block) of the copolymer used (PE0 4 sPCL t7 ).
• the results from the DLS analysis (R H , PDI, ZP) are shown in Table 1.
• the results for the effectivity of encapsulation of temoporfin (%) and the contents of temoporfin (%) in SLNP are shown in Table 2.
• modification lies in weight composition of the lipid phase, where the amount of the 1 -tetradecanol was 50 mg/ml, and in the weight composition of the aqueous phase, where the amount of PEO 45 PCL 34 was 10 mg/ml.
• the modification further consisted in other composition (degree of polymerization of the poly ( ⁇ -caprolactone) block) of the copolymer used (PEO45PCL 3 4).
• the results from the DLS analysis R H , PDI, ZP
• Table 1 The results for the effectivity of encapsulation of temoporfin (%) and the contents of temoporfin (%) in SLNP are shown in Table 2.
• the modification lies in the amount of temoporfin used in the preparation of the nanofomiulation under test, which was 20 mg/ml.
• the results from the DLS analysis (3 ⁇ 4, PDI, ZP) are shown in Table 1.
• the results for the effectivity of encapsulation of temoporfin (%) and the contents of temoporfin (%) in SLNP are shown in Table 2.
• PDI polydisperzity index
• ZP zeta potencial
• Example 12 Formulation according to Example 1 wherein lutecium texafynn was used as photosensitizer.

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