WO2013157973A1 - Genetically modified recombinant fibroblast growth factor (fgf1) conjugated with metal or metal oxide nanoparticles, its dna and amino acid sequences and use - Google Patents

Genetically modified recombinant fibroblast growth factor (fgf1) conjugated with metal or metal oxide nanoparticles, its dna and amino acid sequences and use Download PDF

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WO2013157973A1
WO2013157973A1 PCT/PL2013/050010 PL2013050010W WO2013157973A1 WO 2013157973 A1 WO2013157973 A1 WO 2013157973A1 PL 2013050010 W PL2013050010 W PL 2013050010W WO 2013157973 A1 WO2013157973 A1 WO 2013157973A1
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fgf1
metal
conjugate
growth factor
fibroblast growth
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PCT/PL2013/050010
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French (fr)
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Anna SZLACHCIC
Katarzyna PALA
Piotr JAKIMOWICZ
Małgorzata ZAKRZEWSKA
Jacek OTLEWSKI
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Wrocławskie Centrum Badań Eit+ Sp Z. O.O.
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Publication of WO2013157973A1 publication Critical patent/WO2013157973A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • 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
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • FGF1 fibroblast growth factor
  • the object of this application is to provide a genetically modified recombinant fibroblast growth factor (FGF1) conjugated with metal or metal oxide nanoparticles, preferably gold nanoparticles.
  • FGF1 fibroblast growth factor
  • a potential use of this invention is in medicine, for diagnostic and therapeutic purposes in anticancer therapy.
  • Fibroblast growth factor (FGF) receptors are overexpres sed in many types of cancer, such as breast, prostate and bladder cancers.
  • FGF1 Fibroblast growth factor
  • the specificity of FGF1 interaction with its receptors makes it a potential neoplastic cell targeting protein.
  • a drawback of this growth factor is (in comparison with commonly used antibodies, for example) its low thermodynamic stability and susceptibility to proteolysis resulting in a very short half-life in the body.
  • Introduction of selected point mutations significantly improves the above-mentioned parameters.
  • Introduction of point mutations: Q40P, S47I and H93G led to a stable FGF1 mutant [Zakrzewska et al . , 2005]. Such modification renders the protein resistant to proteolytic degradation and thermal inactivation .
  • cysteine residue at position 117 which is exposed on the protein surface, was mutated to serine (C117S) , to prevent protein binding to the nanoparticle surface via cysteine thiol group present at this position.
  • C117S serine
  • the literature describes an effect of this mutation, which significantly increases protein half-life [Ortega et al . 1991, Culajay et al . , 2000] .
  • Unique physicochemical properties of the nanoparticles result in their increasing use in medicine, particularly in the anticancer therapy. Gold nanoparticles are able to absorb infrared light, and consequently to emitheat. For this reason they are employed in a kind of therapy called photothermal therapy.
  • nanoparticles It is based on introduction of nanoparticles into neoplastic cells and their exposure to near infrared light. It causes a local temperature increase, and therefore weakening or death of cells. It is important for the nanoparticles to target selectively the neoplastic cells, leaving normal cells intact. In order to achieve that, the nanoparticles are conjugated with targeting molecules, such as antibodies, aptamers, etc. Very often during conjugation one face aggregation of the nanoparticles caused by the sensitivity of gold colloids to ionic strength of solution and presence of other substances. It is a serious obstacle to precise tissue application.
  • targeting molecules such as antibodies, aptamers, etc.
  • WO2010114632 describes a method of reversible metal nanoparticle aggregation and deaggregation through the use of polymers (e.g., PEG) and molecules with various functional groups. Such compounds constitute a steric hindrance, enabling to maintain a proper distance between the nanoparticles, and therefore prevent their aggregation.
  • polymers e.g., PEG
  • WO2008119181 suggest similar solution, where gold nanoparticles were coated with nucleotides, which, due to their negative charge, allow for mutual nanoparticle repulsion.
  • the patent literature is reach with documents describing therapeutic use of nanoparticles.
  • WO2006122222 document describes the use of gold and silver nanoparticles for imaging and thermotherapy . In such a case the nanoparticles are coated with various targeting molecules that enable their delivery to selected tissues. Organic compounds and polymers were used to prevent the aggregation, such as CTAB or polyethylene glycol.
  • a first object of the invention is to provide a genetically modified recombinant fibroblast growth factor FGF1 characterised in that it contains a Cys-to-Ser mutation at position 117 in amino acid sequence shown as SEQ ID NO: 2, and which has increased stability and decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle, in comparison to wild type protein.
  • a second object of the invention is to provide a nucleotide sequence of the genetically modified recombinant fibroblast growth factor FGF1 according to first object, shown as SEQ ID NO: 1.
  • a third object of the invention is to provide a modified recombinant fibroblast growth factor characterised in that it contains Cys-to- Ser mutation at position 117 in amino acid sequence shown as SEQ ID NO: 4, which has decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle.
  • a fourth object of the invention is to provide a nucleotide sequence of genetically modified recombinant fibroblast growth factor FGF1 according to third object, shown as SEQ ID NO: 3.
  • a fifth object of the invention is to provide a conjugate of polypeptide with metal or metal oxide nanoparticles, preferably gold nanoparticles (FGFl-Au) characterised in that it contains polypeptide as defined in subject
  • a sixth object of the invention is to provide a conjugate, as defined in the fifth object of the invention, for use in hyperthermia induced by exposition of the conjugate to the near infrared (NIR) light.
  • NIR near infrared
  • the present specification shows FGFl-gold nanoparticle conjugates, designed to specifically orient the protein on the nanoparticle surface and prevent the colloid aggregation.
  • gold nanoparticles may bind both to thiol group located at the end of a flexible linker and to cysteine 117 on the surface of the protein, leading to a nonhomogeneous product with a greater tendency to aggregate.
  • the protein on the surface of gold is oriented in such a way that the region of interaction with FGF receptor is not obstructed by the nanoparticle .
  • FGF1 may then properly interact with receptor and direct nanoparticles specifically to FGF receptor overexpressing cells.
  • Figure 1 shows DLS measurement of gold colloids (la) and FGFl-Au conjugates (lb)
  • Figure 2 shows diagram of FGF1 protein structure with mutations introduced, which enable specific conjugation with gold nanoparticles
  • Figure 3 shows SDS-PAGE gel with applied subsequent eluate FGF1 mutant fractions obtained from Superdex75 column
  • Figure 4 shows denaturation curve for wild type and mutant FGF1
  • Figure 5 shows gold nanoparticle UV-Vis spectra
  • Figure 6 shows a diagram of a gold nanoparticle conjugate with FGF1 mutant
  • Figure 7 shows results of proliferation assay with NIH/3T3 cells treated with FGF1 mutant, FGFl-Au conjugates and gold colloid
  • Figure 8 shows Western Blot analysis of signalling pathways in NIH/3T3 cells activated through incubation with a free FGF1 mutant, FGFl-A
  • Example 1 Gold nanoparticle synthesis Gold colloids were prepared according to standard procedure [Turkevich et al., 1951] . 20 mL solution containing 4 mL 1% (w/v) sodium citrate (Sigma-Aldrich) and 0.08 mL 1% (w/v) tannic acid (Sigma-Aldrich) was added to 80 mL solution containing 1 mL 1% (w/v) aurous acid (Sigma-Aldrich) . The mixture was heated at 60 °C for 20 min on a magnetic stirrer, and then cooled to room temperature.
  • Nanoparticle size was determined with DLS (Dynamic Light Scattering) method using Zetasizer version 6.00 beta 2 (Malvern Instruments). UV-Vis spectra were measured with Cary 300 spectrophotometer (Varian) . DLS measurements indicated that the nanoparticle size was 10 nm ( Figure la)
  • E. coli BL21 (DE3 ) pLysS bacteria strain for recombinant protein overproduction was transformed with modified FGF1 constructs.
  • Culture was grown in a complete LB medium at 37 °C, protein expression was induced with isopropyl ⁇ -D-thiogalactopyranoside (IPTG) with final concentration 0.5 mM at optical density (OD 6 oo ) between 0.6 and 0.8. Then the culture was grown for 4 hours at 37 °C, centrifuged, bacterial pellet was resuspended in buffer containing 20 mM TrisHCl, 0.5 M NaCl, 1 mM EDTA, 0.1 mM PMSF and frozen at -20 °C.
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • OD 6 oo optical density
  • Protein elution was performed using 20 mM TrisHCl buffer, 2 M NaCl, 1 mM EDTA, 1 mM DTT - the protein was released from the bed due to high ionic strength. Protein presence in individual eluate fractions (1 mL) was spectrophotometrically determined basing on absorbance at 280 nm. Purity of the obtained preparation was verified by SDS-PAGE ( Figure 3) and mass spectrometry. Protein nativity was confirmed by fluorescence spectrum analysis. It was based on a fact, that in the natively folded FGF1 molecule tryptophan residue (Trpl07) fluorescence is completely extinguished at 280 nm excitation) .
  • the emission spectrum is characterised by a typical for tryptophan peak at 353 nm. Fluorimetric measurements were performed using Jasco FP-750 spectrofluorimeter, in phosphate buffer (25 mM H 3 P0 4 , pH 7.3), at 21 °C. Emission values were read in the 300-400 nm range, at 280 nm excitation. A cuvette of 1 cm optical path was used and the protein concentration was 4 ⁇ . FGF1 thermal denaturation measurements were performed in phosphate buffer with addition of 0.7 M guanidine hydrochloride (GdmCl) to prevent FGF1 molecule aggregation at increased temperature and two- state denaturing transition. Fluorescence emission increase at 353 nm was measured (excitation at 280 nm) . Temperature change rate was 0.25 °C/min. Optical path was 1 cm, and the protein concentration was 4 ⁇ .
  • Denaturation curves obtained were analysed according to a two-state model (no intermediates during the protein denaturation process) .
  • Denatured and native form fractions were calculated from fluorescence emission changes, and the obtained normalised denaturation curves were analysed with GraFit software (Jandel Scientific Software) ( Figure 4) .
  • Conjugation of FGFl mutant with nanoparticles was based on a ligand exchange on the surface of gold in the presence of a nonionic surfactant. After synthesis the colloid was stabilised with citrate molecules. After nonionic surfactant addition the citrate was displaced and replaced with surfactant molecules. During conjugation n-decyl-p-D-maltoside (DDM) was used in 10 nM concentration, what was a 2-fold excess in relation to critical micelle concentration (CMC) of this compound. The mixture was incubated for 1 hour with nanoparticles at room temperature.
  • DDM critical micelle concentration
  • the FGFl mutant Prior to reaction, the FGFl mutant was reduced with 0.5 mM TCEP and then desalted on PD10 column. The protein was added dropwise to the nanoparticle solution to final concentration 150 ⁇ and incubated overnight at 4 °C. Excess reagents were removed by 4-time centrifugation and the pellet was rinsed with PBS buffer.
  • FIG. 5 shows that the FGFl mutant-coated nanoparticles retain monodispersity (1), while in case of protein conjugates without cysteine 117 mutation the nanoparticles start to aggregate resulting in maximum absorption shift and a broader peak in UV-Vis spectrum ( 2 ) .
  • NIH/3T3 cells were cultured in Quantum 333 medium containing 2% bovine serum.
  • U20S line was cultured in DMEM medium (Dulbecco's modified Eagle's medium) containing 10% fetal calf serum.
  • Stable transfectants overproducing FGFR1 receptor (U20SR1) were obtained through the courtesy of Dr. Ellen M. Haugsten from The Norwegian Radium Hospital .
  • the cells were seeded in 96-well plates (10 4 cells/well), and then the medium was replaced with a serum-free medium. After 24 hours FGFl mutant, FGFl mutant-nanoparticle conjugates and nanoparticles alone, respectively, were added. They were incubated for 48 hours at 37 °C and MTT ( Sigma-Aldrich ) or Alamar Blue (Invitrogen) assays were performed according to the manufacturer's protocol.
  • NIH/3T3 cells cultured for 24 hours in the serum-free medium were stimulated for 15 min with free FGF1 mutant or FGFl-nanoparticle conjugate (FGFl-Au) in 1, 10 and 100 ng/mL protein concentrations in the presence of heparin (10 U/mL) . Then the cells were lysed, sonicated, and the material obtained following SDS-PAGE gel separation was analysed employing Western-Blot technique using anti- phospho-Erkl / 2 , anti-phospho-FGFR, anti-phospho-PLCy, anti-phospho-Akt and anti-y-tubulin antibodies as a control.
  • FGFl-Au FGFl-nanoparticle conjugate
  • FGF1 mutant immobilised on nanoparticles shows the same proliferative activity as free protein ( Figure 7) .
  • Conjugate binds properly with the receptor and stimulates FGFl-activated signalling pathways on the same level as free FGF1 ( Figure 8) .
  • FGF1 mutant fluorescently labelled with Alexa 488 dye was conjugated with nanoparticles according to the protocol described above.
  • U20S and U20SR1 cell lines were cultured on microscopic slides in serum-free medium for 24 hours in 37 °C and then with labelled free FGF1 mutant and labelled FGFl-Au conjugate in 100 ng/mL concentration (based on the protein) for 30 min at 37 °C. After this time slides were fixed in formalin solution and analysed using Zeiss LSM Duo confocal microscope.
  • NIR-induced hyperthermia Experiment was performed on U20SR1 line (stably transfected with FGF1 receptor) and U20S line as a control.
  • Cells were seeded in a 96-well plate in DMEM medium containing 10% fetal calf serum.
  • they were incubated for 1 hour at 37 °C, and then unbound nanoparticles were washed off.
  • the respective wells were irradiated with 808 nm laser (1.2A, Optel Instruments) for 10 and 20 min.
  • hyperthermia induction cells were incubated at 37 °C for 12 hours. The viability was verified by MTT (Sigma-Aldrich) assay according to the manufacturer's protocol. The experiment was made in quadruplicate.

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Abstract

The object of the invention is to provide a genetically modified recombinant fibroblast growth factor 1 (FGF1) characterised in that it contains Cys-to-Ser mutation at position (117) in amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 3, and which has decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle. The next object of the invention is to provide a conjugate of metal or metal oxide nanoparticles (preferably gold nanoparticles) with the said polypeptide, and its use in induction of hyperthermia caused by exposition of conjugate to near infrared (NIR) light.

Description

Genetically modified recombinant fibroblast growth factor (FGF1) conjugated with metal or metal oxide nanoparticles, its DNA and amino acid sequences and use.
The object of this application is to provide a genetically modified recombinant fibroblast growth factor (FGF1) conjugated with metal or metal oxide nanoparticles, preferably gold nanoparticles. A potential use of this invention is in medicine, for diagnostic and therapeutic purposes in anticancer therapy.
Fibroblast growth factor (FGF) receptors are overexpres sed in many types of cancer, such as breast, prostate and bladder cancers. The specificity of FGF1 interaction with its receptors makes it a potential neoplastic cell targeting protein. However, a drawback of this growth factor is (in comparison with commonly used antibodies, for example) its low thermodynamic stability and susceptibility to proteolysis resulting in a very short half-life in the body. Introduction of selected point mutations significantly improves the above-mentioned parameters. Introduction of point mutations: Q40P, S47I and H93G led to a stable FGF1 mutant [Zakrzewska et al . , 2005]. Such modification renders the protein resistant to proteolytic degradation and thermal inactivation . Furthermore, cysteine residue at position 117, which is exposed on the protein surface, was mutated to serine (C117S) , to prevent protein binding to the nanoparticle surface via cysteine thiol group present at this position. The literature describes an effect of this mutation, which significantly increases protein half-life [Ortega et al . 1991, Culajay et al . , 2000] . Unique physicochemical properties of the nanoparticles result in their increasing use in medicine, particularly in the anticancer therapy. Gold nanoparticles are able to absorb infrared light, and consequently to emitheat. For this reason they are employed in a kind of therapy called photothermal therapy. It is based on introduction of nanoparticles into neoplastic cells and their exposure to near infrared light. It causes a local temperature increase, and therefore weakening or death of cells. It is important for the nanoparticles to target selectively the neoplastic cells, leaving normal cells intact. In order to achieve that, the nanoparticles are conjugated with targeting molecules, such as antibodies, aptamers, etc. Very often during conjugation one face aggregation of the nanoparticles caused by the sensitivity of gold colloids to ionic strength of solution and presence of other substances. It is a serious obstacle to precise tissue application. WO2010114632 document describes a method of reversible metal nanoparticle aggregation and deaggregation through the use of polymers (e.g., PEG) and molecules with various functional groups. Such compounds constitute a steric hindrance, enabling to maintain a proper distance between the nanoparticles, and therefore prevent their aggregation. The authors of WO2008119181 suggest similar solution, where gold nanoparticles were coated with nucleotides, which, due to their negative charge, allow for mutual nanoparticle repulsion. The patent literature is reach with documents describing therapeutic use of nanoparticles. WO2006122222 document describes the use of gold and silver nanoparticles for imaging and thermotherapy . In such a case the nanoparticles are coated with various targeting molecules that enable their delivery to selected tissues. Organic compounds and polymers were used to prevent the aggregation, such as CTAB or polyethylene glycol.
However, there are some limitations to polymer and organic compound use as anti-aggregation agents. Binding of protein or other biologically active molecule to nanoparticles causes disturbances in interactions between individual particles resulting in nanoparticle precipitation. Therefore, there is a search for methods to bind proteins to gold nanoparticles, which would provide a reduced nanoparticle aggregation in comparison to approaches of the prior art. Unexpectedly, the above-mentioned problem was resolved by the present invention. A first object of the invention is to provide a genetically modified recombinant fibroblast growth factor FGF1 characterised in that it contains a Cys-to-Ser mutation at position 117 in amino acid sequence shown as SEQ ID NO: 2, and which has increased stability and decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle, in comparison to wild type protein.
A second object of the invention is to provide a nucleotide sequence of the genetically modified recombinant fibroblast growth factor FGF1 according to first object, shown as SEQ ID NO: 1.
A third object of the invention is to provide a modified recombinant fibroblast growth factor characterised in that it contains Cys-to- Ser mutation at position 117 in amino acid sequence shown as SEQ ID NO: 4, which has decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle.
A fourth object of the invention is to provide a nucleotide sequence of genetically modified recombinant fibroblast growth factor FGF1 according to third object, shown as SEQ ID NO: 3.
A fifth object of the invention is to provide a conjugate of polypeptide with metal or metal oxide nanoparticles, preferably gold nanoparticles (FGFl-Au) characterised in that it contains polypeptide as defined in subject
A sixth object of the invention is to provide a conjugate, as defined in the fifth object of the invention, for use in hyperthermia induced by exposition of the conjugate to the near infrared (NIR) light. The present specification shows FGFl-gold nanoparticle conjugates, designed to specifically orient the protein on the nanoparticle surface and prevent the colloid aggregation.
A specific conjugation with nanoparticles, via cysteine thiol group, was obtained by removal of cysteine at position 117, which is naturally present in the protein and exposed on its surface - it was replaced with a serine residue (C117S mutation) . Furthermore, at the protein N-terminus there was a flexible linker introduced with a cysteine terminal group. In the described mutant there is only one exposed cysteine - at the N-terminus of the protein, which enables a direct binding of the growth factor to gold and does not disturb the protein structure. In case when the cysteine residue 117 is not mutated to serine, gold nanoparticles may bind both to thiol group located at the end of a flexible linker and to cysteine 117 on the surface of the protein, leading to a nonhomogeneous product with a greater tendency to aggregate. Moreover, thanks to the introduction of C117S mutation the protein on the surface of gold is oriented in such a way that the region of interaction with FGF receptor is not obstructed by the nanoparticle . FGF1 may then properly interact with receptor and direct nanoparticles specifically to FGF receptor overexpressing cells.
Example embodiments of the invention are presented below on the drawings, where Figure 1 shows DLS measurement of gold colloids (la) and FGFl-Au conjugates (lb), Figure 2 shows diagram of FGF1 protein structure with mutations introduced, which enable specific conjugation with gold nanoparticles, Figure 3 shows SDS-PAGE gel with applied subsequent eluate FGF1 mutant fractions obtained from Superdex75 column, Figure 4 shows denaturation curve for wild type and mutant FGF1, Figure 5 shows gold nanoparticle UV-Vis spectra; spectrum 1 - FGF1 mutant conjugates, 2 - conjugates of nanoparticles with stable FGF1 mutant without C117S substitution, Figure 6 shows a diagram of a gold nanoparticle conjugate with FGF1 mutant, Figure 7 shows results of proliferation assay with NIH/3T3 cells treated with FGF1 mutant, FGFl-Au conjugates and gold colloid, Figure 8 shows Western Blot analysis of signalling pathways in NIH/3T3 cells activated through incubation with a free FGF1 mutant, FGFl-Au and gold colloids, Figure 9 shows confocal microscopy images of specific labelled mutant FGF1 and FGFl-Au conjugate internalisation in FGFR1 receptor-positive cells; 1 - Alexa 488-FGF1, 2 - nuclei stained with DAPI dye, 3 - superimposition of image 1 and 2; a - U20SR1 cells, b - U20S cells, and Figure 10 shows the viability of U20SR1 (a) and U20S cells (b) treated with FGFl-Au conjugates, AuNP nanoparticles and PBS buffer, and irradiated with NIR.
Example 1 Gold nanoparticle synthesis Gold colloids were prepared according to standard procedure [Turkevich et al., 1951] . 20 mL solution containing 4 mL 1% (w/v) sodium citrate (Sigma-Aldrich) and 0.08 mL 1% (w/v) tannic acid (Sigma-Aldrich) was added to 80 mL solution containing 1 mL 1% (w/v) aurous acid (Sigma-Aldrich) . The mixture was heated at 60 °C for 20 min on a magnetic stirrer, and then cooled to room temperature. Nanoparticle size was determined with DLS (Dynamic Light Scattering) method using Zetasizer version 6.00 beta 2 (Malvern Instruments). UV-Vis spectra were measured with Cary 300 spectrophotometer (Varian) . DLS measurements indicated that the nanoparticle size was 10 nm (Figure la)
Example 2
Overproduction, purification and biophysical characterization of FGF1 mutant In the sequence encoding human FGF1, which was cloned into pET-3c vector, exposed cysteine was substituted with serine (C117S) ; it contained also a four amino acid (Cys-Gly-Gly-Gly ) insertion on the protein N-terminus introduced by QuikChange site-directed mutagenesis kit (Stratagene) . Obtained constructs were sequenced to confirm the presence of introduced mutations. Constructs with proper sequence were propagated in E. coli DH10 strain and purified. Purity of DNA preparations was confirmed by agarose gel electrophoresis.
E. coli BL21 (DE3 ) pLysS bacteria strain for recombinant protein overproduction was transformed with modified FGF1 constructs. Culture was grown in a complete LB medium at 37 °C, protein expression was induced with isopropyl β-D-thiogalactopyranoside (IPTG) with final concentration 0.5 mM at optical density (OD6oo ) between 0.6 and 0.8. Then the culture was grown for 4 hours at 37 °C, centrifuged, bacterial pellet was resuspended in buffer containing 20 mM TrisHCl, 0.5 M NaCl, 1 mM EDTA, 0.1 mM PMSF and frozen at -20 °C.
After thawing bacterial pellets were sonicated at 4 °C and centrifuged (14000 g, 4 °C, 1 hour) . After centrifugation, supernatant constituting the soluble protein fraction was subjected to binding with Heparin-Sepharose bed pre-equilibrated with 20 mM TrisHCl, 0.5 M NaCl, 1 mM EDTA, 1 mM DTT . Next, the bed was rinsed with cooled buffer of above composition and successively with buffer containing 20 mM TrisHCl, 0.7 M NaCl, 1 mM EDTA, 1 mM DTT. Protein elution was performed using 20 mM TrisHCl buffer, 2 M NaCl, 1 mM EDTA, 1 mM DTT - the protein was released from the bed due to high ionic strength. Protein presence in individual eluate fractions (1 mL) was spectrophotometrically determined basing on absorbance at 280 nm. Purity of the obtained preparation was verified by SDS-PAGE (Figure 3) and mass spectrometry. Protein nativity was confirmed by fluorescence spectrum analysis. It was based on a fact, that in the natively folded FGF1 molecule tryptophan residue (Trpl07) fluorescence is completely extinguished at 280 nm excitation) . In a denatured state the emission spectrum is characterised by a typical for tryptophan peak at 353 nm. Fluorimetric measurements were performed using Jasco FP-750 spectrofluorimeter, in phosphate buffer (25 mM H3P04, pH 7.3), at 21 °C. Emission values were read in the 300-400 nm range, at 280 nm excitation. A cuvette of 1 cm optical path was used and the protein concentration was 4 μΜ. FGF1 thermal denaturation measurements were performed in phosphate buffer with addition of 0.7 M guanidine hydrochloride (GdmCl) to prevent FGF1 molecule aggregation at increased temperature and two- state denaturing transition. Fluorescence emission increase at 353 nm was measured (excitation at 280 nm) . Temperature change rate was 0.25 °C/min. Optical path was 1 cm, and the protein concentration was 4 μΜ.
Denaturation curves obtained were analysed according to a two-state model (no intermediates during the protein denaturation process) . Denatured and native form fractions were calculated from fluorescence emission changes, and the obtained normalised denaturation curves were analysed with GraFit software (Jandel Scientific Software) (Figure 4) .
Denaturation measurements were carried out simultaneously for mutant and wild type (WT) FGF1. Example 3 Conjugation of FGFl mutant with gold nanoparticles
Conjugation of FGFl mutant with nanoparticles was based on a ligand exchange on the surface of gold in the presence of a nonionic surfactant. After synthesis the colloid was stabilised with citrate molecules. After nonionic surfactant addition the citrate was displaced and replaced with surfactant molecules. During conjugation n-decyl-p-D-maltoside (DDM) was used in 10 nM concentration, what was a 2-fold excess in relation to critical micelle concentration (CMC) of this compound. The mixture was incubated for 1 hour with nanoparticles at room temperature.
Prior to reaction, the FGFl mutant was reduced with 0.5 mM TCEP and then desalted on PD10 column. The protein was added dropwise to the nanoparticle solution to final concentration 150 μΜ and incubated overnight at 4 °C. Excess reagents were removed by 4-time centrifugation and the pellet was rinsed with PBS buffer.
Colloid homogeneity was analysed by spectrophotometry in UV-Vis range. Figure 5 shows that the FGFl mutant-coated nanoparticles retain monodispersity (1), while in case of protein conjugates without cysteine 117 mutation the nanoparticles start to aggregate resulting in maximum absorption shift and a broader peak in UV-Vis spectrum ( 2 ) .
Example 4
Proliferation assays
NIH/3T3 cells were cultured in Quantum 333 medium containing 2% bovine serum. U20S line was cultured in DMEM medium (Dulbecco's modified Eagle's medium) containing 10% fetal calf serum. Stable transfectants overproducing FGFR1 receptor (U20SR1) were obtained through the courtesy of Dr. Ellen M. Haugsten from The Norwegian Radium Hospital . The cells were seeded in 96-well plates (104 cells/well), and then the medium was replaced with a serum-free medium. After 24 hours FGFl mutant, FGFl mutant-nanoparticle conjugates and nanoparticles alone, respectively, were added. They were incubated for 48 hours at 37 °C and MTT ( Sigma-Aldrich ) or Alamar Blue (Invitrogen) assays were performed according to the manufacturer's protocol.
Cell signal tranduction tests
NIH/3T3 cells cultured for 24 hours in the serum-free medium were stimulated for 15 min with free FGF1 mutant or FGFl-nanoparticle conjugate (FGFl-Au) in 1, 10 and 100 ng/mL protein concentrations in the presence of heparin (10 U/mL) . Then the cells were lysed, sonicated, and the material obtained following SDS-PAGE gel separation was analysed employing Western-Blot technique using anti- phospho-Erkl / 2 , anti-phospho-FGFR, anti-phospho-PLCy, anti-phospho- Akt and anti-y-tubulin antibodies as a control.
Proliferation assays (MTT and Alamar Blue) indicate, that FGF1 mutant immobilised on nanoparticles shows the same proliferative activity as free protein (Figure 7) . Conjugate binds properly with the receptor and stimulates FGFl-activated signalling pathways on the same level as free FGF1 (Figure 8) .
Example 5
Specific FGFl-Au conjugate internalisation
Experiment confirming specific FGFl-Au conjugate internalisation in FGFR1 receptor overexpressing cells was performed on U20SR1 lines stable transfected with FGFR1 receptor gene. U20S line not expressing any of the receptor isoforms was used as a control.
FGF1 mutant fluorescently labelled with Alexa 488 dye was conjugated with nanoparticles according to the protocol described above. U20S and U20SR1 cell lines were cultured on microscopic slides in serum-free medium for 24 hours in 37 °C and then with labelled free FGF1 mutant and labelled FGFl-Au conjugate in 100 ng/mL concentration (based on the protein) for 30 min at 37 °C. After this time slides were fixed in formalin solution and analysed using Zeiss LSM Duo confocal microscope.
Confocal microscopy images show that only in case of U20SR1 cells Alexa 488 labelled FGF1 mutant and FGFl-Au conjugate are internalised (Figure 9) . In case of U20S control cells neither FGF1 as such nor nanoparticle-protein conjugates were internalised. Because the only difference between the selected lines was the presence or absence of FGFR1 receptor the obtained result indicates a specific receptor-dependent internalisation . It indicates also that protein present on the nanoparticle surface is properly folded and fully active.
Example 6
NIR-induced hyperthermia Experiment was performed on U20SR1 line (stably transfected with FGF1 receptor) and U20S line as a control. Cells were seeded in a 96-well plate in DMEM medium containing 10% fetal calf serum. To appropriate wells FGFl-Au conjugates, and protein uncoated colloids and PBS buffer as controls, were added. Next, they were incubated for 1 hour at 37 °C, and then unbound nanoparticles were washed off. The respective wells were irradiated with 808 nm laser (1.2A, Optel Instruments) for 10 and 20 min. After hyperthermia induction cells were incubated at 37 °C for 12 hours. The viability was verified by MTT (Sigma-Aldrich) assay according to the manufacturer's protocol. The experiment was made in quadruplicate.
Decreased cell viability was observed only in case of U20SR1 cells treated with FGFl-Au conjugates (Figure 10a) . This suggests that the nanoparticles were specifically internalised by FGFR1 receptor- positive cells. In case of controls, to which protein uncoated nanoparticles and PBS buffer were added, no changes of viability resulting from NIR irradiation (Figure 10b) were observed.

Claims

13 Claims
1. Genetically modified recombinant fibroblast growth factor FGF1 characterised in that it contains a Cys-to-Ser mutation at position 117 in amino acid sequence shown as SEQ ID NO: 2, and which has increased stability and decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle, in comparison to wild type protein.
2. Nucleotide sequence of the genetically modified recombinant fibroblast growth factor FGF1 according to claim 1, shown as SEQ ID
NO: 1.
3. Modified recombinant fibroblast growth factor characterised in that it contains Cys-to-Ser mutation at position 117 in amino acid sequence shown as SEQ ID NO: 4, which has decreased aggregation properties in conjugate with metal or metal oxide nanoparticle, preferably gold nanoparticle.
4. Nucleotide sequence of genetically modified recombinant fibroblast growth factor FGF1 according to claim 3, shown as SEQ ID
NO: 3.
5. Conjugate of polypeptide with metal or metal oxide nanoparticles, preferably gold nanoparticles (FGFl-Au) characterised in that it contains polypeptide according to claims 1 or 3.
6. Conjugate according to claim 5 for use in induction of hyperthermia caused by conjugate exposition to near infrared (NIR) light .
PCT/PL2013/050010 2012-04-21 2013-04-19 Genetically modified recombinant fibroblast growth factor (fgf1) conjugated with metal or metal oxide nanoparticles, its dna and amino acid sequences and use WO2013157973A1 (en)

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JOSHUA M. KOGOT ET AL: "Analysis of the Dynamics of Assembly and Structural Impact for a Histidine Tagged FGF1-1.5 nm Au Nanoparticle Bioconjugate", BIOCONJUGATE CHEMISTRY, vol. 20, no. 11, 18 November 2009 (2009-11-18), pages 2106 - 2113, XP055079340, ISSN: 1043-1802, DOI: 10.1021/bc900224d *
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CN113194980A (en) * 2018-10-09 2021-07-30 利兰斯坦福初级大学董事会 Engineered fibroblast growth factor variants as receptor antagonists

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