Supramolecular hacky sacks (SHS), method of synthesis and applications thereof
10106572 ยท 2018-10-23
Assignee
Inventors
Cpc classification
A61K47/6949
HUMAN NECESSITIES
A61K47/549
HUMAN NECESSITIES
International classification
Abstract
The invention is directed to small molecules that self-assemble hierarchically to form NPs termed SHS (due to their architectural features). These SHS are composed by precise supramolecules known as Supramolecular G-Quadruplexes (SGQs) which are formed when amphiphilic guanosine (G) derivatives that self-assemble in presence of salt by non-covalent interactions. The resulting SGQs are made of amphiphilic guanosine (G) subunits (with precisely eight subunits at neutral pH (pH>5.7) or sixteen subunits at acidic pH (pH<5.7)). The SGQs are responsive entities that further self-assemble upon an external stimulus, such as an increase in temperature or a change in pH, leading to the formation of the aforementioned SHS.
Claims
1. A method of synthesizing a cluster of self-arranged supramolecular structures comprising: mixing KI salts with a phosphate buffer solution (PBS) and molecules with the chemical structure (I), ##STR00001## allowing dissolution of said molecules in the (PBS) effectively forming supramolecular structures; removing said supramolecular structures from said mixture and diluting said removed supramolecular structures in PBS without KI salt to lower the ionic strength, effectively fixing together said supramolecular structures; and inducing the fixed supramolecular structures to form a cluster of self-arranged supramolecular structures.
2. The method of claim 1, wherein said KI salts and said molecules are mixed together prior to mixing with said (PBS).
3. The method of claim 1, wherein said mixture is cooled for an amount of time sufficient to allow complete dissolution of said molecules in the (PBS).
4. The method of claim 1, wherein said mixture has a neutral pH.
5. The method of claim 1, wherein said mixture has an acidic pH.
6. The method of claim 4, wherein each of said supramolecular structures comprises 8 subunits of said molecules.
7. The method of claim 5, wherein each of said supramolecular structures comprises 16 subunits of said molecules.
8. The method of claim 1, wherein said supramolecular structures are induced to form said cluster of self-arranged supramolecular structures by subjecting said supramolecular structures to a temperature equal to or greater than a transition temperature.
9. The method of claim 8, wherein the pH of said mixture is neutral.
10. The method of claim 8, wherein the pH of said mixture is acidic.
11. The method of claim 8, wherein said transition temperature is about 8-15 C.
12. The method of claim 8, wherein said transition temperature is about 42-50.5 C.
13. The method of claim 8, wherein said transition temperature is about 4-5.5 C.
14. The method of claim 8, wherein said transition temperature is about 29-59.3 C.
15. The method of claim 1, wherein said mixture is cooled at a temperature below said transition temperature.
16. A method of encapsulating a guest material inside a cluster of self-arranged supramolecular structures, said method comprising: mixing a guest material with KI salts, phosphate buffer solution (PBS) and clusters of self-arranged supramolecular structures, wherein said clusters of self-arranged supramolecular structures are diluted in PBS in the absence of KI salt for fixing said clusters of self-arranged supramolecular structures by lowering the ionic strength prior to mixing with said guest material, each supramolecular structure comprises a plurality of molecules of the chemical structure: ##STR00002## and agitating said mixture for an amount of time sufficient to allow said guest material to diffuse through said clusters of fixed self-arranged supramolecular structures, effectively encapsulating said guest material inside the clusters of fixed self-arranged supramolecular structures.
17. The method of claim 16, wherein said guest material is in solid state.
18. The method of claim 16, wherein said guest material is in liquid state.
19. The method of claim 16, wherein said guest material is a polymer.
20. The method of claim 16, wherein said guest material is a protein.
21. The method of claim 16, wherein said guest material is a molecule.
22. The method of claim 16, wherein said guest material is nucleic acid.
23. A method of releasing a guest material encapsulated inside a cluster of self-arranged supramolecular structures, said method comprising: modifying a cluster of self-assembled supramolecular structures encapsulating a guest material, said cluster comprises supramolecular structures having a plurality of molecules of the chemical structure: ##STR00003## wherein said cluster is modified by diluting said cluster in PBS in the absence of KI salt to lower the ionic strength, effectively fixing together said cluster of self-arranged supramolecular structures to allow controlled release of said guest material.
24. The method of claim 23, wherein said guest material is in solid state.
25. The method of claim 23, wherein said guest material is in liquid state.
26. The method of claim 23, wherein said guest material is a polymer.
27. The method of claim 23, wherein said guest material is a protein.
28. The method of claim 23, wherein said guest material is a molecule.
29. The method of claim 23, wherein said guest material is nucleic acid.
30. A method of releasing a guest material encapsulated inside a cluster of self-arranged supramolecular structures, said method comprising: modifying a cluster of fixed self-assembled supramolecular structures encapsulating a guest material, said cluster comprises supramolecular structures having a plurality of molecules of the chemical structure: ##STR00004## that have been previously diluted in PBS in the absence of KI salt to lower the ionic strength, wherein said cluster is modified by subjecting said cluster to a surrounding environment having an acidic pH level, effectively disassembling said supramolecular structures to allow immediate release of said guest material.
31. The method of claim 30, wherein said guest material is in solid state.
32. The method of claim 30, wherein said guest material is in liquid state.
33. The method of claim 30, wherein said guest material is a polymer.
34. The method of claim 30, wherein said guest material is a protein.
35. The method of claim 30, wherein said guest material is a molecule.
36. The method of claim 30, wherein said guest material is nucleic acid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Further features and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying figure showing illustrative embodiments of the invention, in which:
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DETAILED DESCRIPTION OF THE INVENTION
(38) The present invention is directed to 8ArG derivatives that self-assemble in thermoresponsive SGQs and that also respond to pH as an alternative stimulus.
(39) Procedure for the Preparation of ImAGhD2OH, ImAGpD2OH and ImAG Standard
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(41) The same procedure explained above was applied with ImAGpD2OH (with a molecular structure as shown in
(42) During the synthesis of derivatives ImAGhD2OH and ImAGpD2OH, an ImAG std (with a molecular structure as shown in
(43) Procedure to Induce Formation of Supramolecular Structures
(44) One important aspect of the invention is the self-assembly properties of the small molecule of ImAG in the presence of salt (e.g., KI) at a certain pH as seen on
(45) Solution Preparation
(46) Solutions of 0.650 mL of ImAGhD2OH were prepared at 5 mM with 4 M KI. In the case of ImAGpD2OH, the solutions prepared were 5 mM with 2 M KI. By following this procedure the following ratio is recommended between ImAGhD2OH (or ImAGpD2OH) with KI salt: 0-10 mM of ImAGhD2OH (or ImAGpD2OH) with 2-4 M KI. To dissolve the solids it was prepared a 0.1 M sodium acetate buffer for pH values of 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9. For pH 6-7, PBS from Fisher Scientific was used. The samples were put in the refrigerator (10 C.) overnight (12 h) for equilibration. This sample concentration was used for turbidity experiments, DSC, and DLS experiments. The concentrations of the resulting assemblies of SGQ and f-SHS were calculated in terms of the molecular weights of monomeric 8ArG derivative ImAGpD2OH or ImAGhD2OH.
(47) Self-Assembly NMR Studies
(48) To confirm that the self-assembly of ImAGhD2OH (or ImAGpD2OH) was successful after the solid entered in solution, the supramolecular G-quadruplex assembly (SGQ or supramolecular structure) assembly by .sup.1H NMR was confirmed. Self-assembly studies were carried out using a Bruker AV-500 NMR spectrometer, equipped with a 5 mm BBO probe. In water, a conventional 1D presaturation pulse sequence with the excitation pulse set over the water peak at 4.7 ppm was used. A standard proton sequence was used for experiments in D.sub.2O. Self-assembly studies were performed, for example, using a 10 mM solution of ImAGhD2OH in 650 L of H.sub.2O were 10% of standard (Sodium 3-(trimethylsilyl)propionate-2,2,3,3-d.sub.4 from Aldrich) was used in D.sub.2O (9:1 H.sub.2O: D.sub.2O, 4 M KI, 298 K). The same conditions were used for ImAGpD2OH, but with 2 M of KI.
(49) To study the resulting assembly of compounds ImAGhD2OH and ImAGpD2OH, .sup.1H NMR experiments were performed to confirm the signature peaks in the N.sub.1H region that correspond to a nanoglobular SGQ (supramolecular structure) composed of eight subunits at pH 7 (oSGQ), but at pH 5 the molecularity of the SGQ is composed of sixteen subunits (hSGQ) as shown in
(50) Procedure to Measure a Transition pH that Induces Formation of Cluster of Self-Arranged Supramolecular Structures
(51) Before the imidazole modification of ImAGpD2OH and ImAGhD2OH, chalcone imidazole standard derivative (E)-3-(1-methyl-1H-imidazol-2-yl)-1-phenylprop-2-en-1-one (ImAG std) was synthesized by the same aldol condensation method to obtain an estimation of the pKa by measuring the absorbance at different pHs in a Varian UV-visible spectrometer, Model Cary Bio-100 as shown in
(52) Also, isothermal turbidity experiments of ImAGhD2OH (transmittance at 650 nm, 26 C.) confirmed a pH-responsive behavior revealing a transition pH around 5.7 at 65% of the transmittance as shown in
(53) Procedure of Formation of SHS by Temperature or pH
(54) One aspect of the present invention is directed to a method of constructing SHS microparticles, which as previously explained, are composed of SGQs, which in turn are composed of ImAG (ImAGpD2OH or ImAGhD2OH) small molecules. The construction process of the SHS starts by the self-assembly of ImAG molecules in the presence of a suitable salt (e.g., KI) leading to the formation of SGQs that are composed of precisely eight (8) ImAG subunits (at pH 7) or sixteen (16) ImAG subunits (at pH 5). The SGQs in turn can be triggered to further self-assembly to form the SHS via heating the sample above a threshold temperature or via a change in pH (e.g., acidity of the medium) as illustrated in
(55) Procedure to Measure a Transition Temperature that Induces Formation of Cluster of Self-Arranged Supramolecular Structures
(56) Turbidimetry Studies (T.sub.cp)
(57) The effect of the imidazole moiety in ImAGhD2OH is not only limited to a pH-response behavior, the thermoresponsive behavior is maintained in the SGQ (supramolecular structure), thus exhibiting dual-responsive behavior as shown in
(58) As shown in
(59) b) Differential Scanning Calorimetry (DSC) Experiments (T.sub.t)
(60) These DSC experiments support the performed turbidity experiments and with additional thermodynamic parameters provide an insight into the enthalpy of the SGQs assemblies at different pHs as shown in
(61) The endotherms for oSGQ at pH 7 show a T.sub.t of 5.4 C., but for hSGQ at pH 5 the T.sub.t is 59.3 C. as seen in Table 1 below. Like in the turbidity experiments, there's a significant difference in T.sub.t (T.sub.t53.9 C.) between SGQs oSGQ and hSGQ that can be related to the protonation of the imidazole moiety in hSGQ at pH<6. The effect of the imidazole protonation in the SGQ (supramolecular structure) assembly is reflected in the T.sub.m of hSGQ and oSGQ, which are 72.7 C. and 42.6 C., respectively, with oSGQ being the least thermally stable assembly as shown in Table 1 below. The evident discrepancy between the onset of turbidity (T.sub.cp) and T.sub.t of the maximum DSC peak in hSGQ was observed in previous reported SGQs and micellar telechelic poly(N-isopropylacrylamide) (PNIPAM) reported by Winnik and co-workers. This phenomenon is related to an athermal association process between the nanoglobular SGQ assemblies by non-covalent interactions of the hydroxyl groups of the periphery with the water molecules. The water-mediated association process is reflected in the SHS microglobules with sizes large enough to scatter light during the phase transition (measured by the T.sub.cp). However, the separation of water molecules of hydration shell of the SGQ to the bulk is below the detection of the calorimeter resulting in higher T.sub.t values that are related to the energy required to expel associated water molecules. The enthalpy (H.sub.t) of this phase transition process is related to the number of water molecules associated with the hydration shell of the SGQ microglobules. This is presented in the low amount of water molecules associated in oSGQ at pH 7 with a H.sub.t of 0.078 kcal/mol in comparison with the high magnitude value (H.sub.t) in hSGQ at pH 5. Interestingly, at pH 5.5 (near the transition pH 5.7), there is a significant change in the magnitude of H.sub.t to 146 kcal/mol showing the effect of the number of water molecules associated during the assembly process between oSGQ and hSGQ.
(62) Table 1 shows the thermodynamic parameters for the LCST phenomenon (T.sub.t) and for the melting of ImAGhD2OH at different pH values in aqueous solution as determined by DSC. The G.sub.cal and S.sub.cal values shown in this table correspond to those calculated using the values of T.sub.t, T.sub.m, and H.sub.cal from the instrument. All G values were calculated at a temperature of 20 C.
(63) TABLE-US-00001 TABLE 1 T.sub.t G.sub.T: H.sub.Tt S.sub.Tt T.sub.m G.sub.Tm H.sub.Tm S.sub.Tm pH ( C.) (kcal/mol) (kcal/mol) (kcal/mol .Math. C.) ( C.) (kcal/mol) (kcal/mol) (kcal/mol .Math. C.) 5.0 59.26 30.67 46 13 0.778 72.72 46.64 64.32 0.884 5.5 54.00 91.92 146.0 2.704 68.64 44.34 62.58 0.912 6:0 5.702 0.309 0.123 0.0216 41.12 5.76 11.20 0.272 6.5 4.938 0.160 0.0522 0.0106 37.32 5.48 11.80 0.316 7.0 5.449 0.210 0.0784 0.0144 42.64 8.18 15.42 0.362
(64) Differential Scanning calorimetry (DSC) analyses were performed on a VP-DSC Micro-Calorimeter from MicroCal and Origin (v.7) was used for data processing. For the experiment, the buffer was first degasified and the experiment was carried out as a blank in both sample and reference cell. Then, the sample was degasified, and the blank buffer in the sample cell to be substituted with sample was removed. Afterwards, the experiment was performed with a temperature ramp of 0 C.-80 C. for 4 cycles.
(65) LCST Modulation in pH-Responsive SGQ
(66) The present invention provides a pH and thermo responsive SGQ that can form a precise assembly hSGQ when pH<6 and oSGQ when pH>6 by the incorporation of the imidazole group in the C8 position of the guanine. But, it is know from previous reported SGQ derivatives that modulation of LCST could be achieved by changing the three-dimensional distribution of hydrophobic patches in the form of methylenes in the periphery of the ribose chains of the SGQ. For that reason, ImAGpD2OH was synthesized, which exhibits the same self-assembly properties presented in ImAGhD2OH by forming oSGQ at pH 7 and hSGQ at pH 5 as shown in
(67) TABLE-US-00002 TABLE 2 T.sub.t G.sub.T: H.sub.Tt S.sub.Tt T.sub.m G.sub.Tm H.sub.Tm S.sub.Tm pH ( C.) (kcal/mol) (kcal/mol) (kcal/mol .Math. C.) ( C.) (kcal/mol) (kcal/mol) (kcal/mol .Math. C.) 5.0 50.45 5.07 8.41 0.167 70.52 40.85 57.01 0.808 7.0 8.631 5.74 4.36 0.505 56.11 65.20 101.3 1.805
(68) Method to Fix Cluster of Self-Arranged Supramolecular Structures
(69) The present invention also provides a method for stabilizing the SHS via a fixing protocol in which the resulting f-SHS (the prefix f- is added to SHS to denote a fixed SHS) tolerate a variety of physical manipulations like dilutions, pipetting, freeze-drying, transfers between different media (e.g., cell growth serum). The term fixing is used herein as an analogy to the process of fixing cells in cell biology (e.g., using aldehydes as cross-linking agents) to preserve its morphological features for microscopy studies. This method consists in decreasing the ionic strength of the solution from molar to millimolar range without disturbing the globular morphology of the SHS as illustrated in
(70) The fixing protocol requires no additional stabilizing substances or further covalent modifications like cross-linking agents. This protocol also serves to rinse the f-SHS after the encapsulation of a desired guest (e.g., molecules, polymers, proteins and nucleic acids) with negligible loss of such guest (as determined by evaluating the concentration of guests in the surrounding media). The rinsing of the excess guest and dilution of the f-SHS enable the use of these particles under conditions suitable for biomedical applications as described below.
(71) The concentrations of the resulting assemblies of SGQ and f-SHS were calculated in terms of the molecular weights of monomeric 8ArG derivative ImAGpD2OH (or ImAGhD2OH). For the purpose of the following explanations, the specification will refer to ImAGpD2OH, but the same procedures apply to ImAGhD2OH. For the fixed SHS (f-SHS) concentrations, 0.1 mL of the SHS colloid was diluted at 40 C. formed by LCST in 1.57 mL of PBS (1 at pH 7.4) to obtain a resulting concentration of 0.303 mM f-SHS and 121 mM KI. By lowering the ionic strength with this dilution process, kinetically stable versions of the SHS were isolated. In other words, by using this method, f-SHS were obtained. One important advantage of this fixing process is that the f-SHS can be diluted from molar to millimolar range while preserving their integrity and shape without the need of covalent crosslinking agents or further modifications to increase the stability.
(72) The fixing process according to the present invention will be explained in detail. The vial with the SHS is removed from the fridge (10 C.-0 C.) and transferred to ice. If the sample is frozen, the sample is agitated until the ice get melted and then transferred into ice. Afterwards, the vial with the SHS is removed from the ice and placed in a water bath at 40 C. (temperature used have to be equal or above the T.sub.cp (or T.sub.t) of the SGQ in solution) for about 1 min under manual stirring until the solution becomes cloudy (SHS colloid). With a micropipette, 250 L is taken from the SHS vial and transferred into a first vial that already contains 750 L of PBS (total volume 1.00 mL). The first vial is gently agitated for 1 minute to homogenize the solution. Finally, the SHS vial is returned immediately to the ice and the sample can be returned to the fridge.
(73) If other concentrations of f-SHS want to be used, the following dilutions can be used. With a micropipette, 80 L is taken from the first vial and transferred into a second vial that contains 120 L of PBS (total volume of 200 L).
(74) The second vial is gently agitated for 1 minute to homogenize the solution. This process is also carried out for the other samples. With a micropipette, 32 L is taken from the first vial and transferred into a third vial, which contains 168 L of PBS (total volume of 200 L). The third vial is gently agitated for 1 minute to homogenize the solution. With a micropipette, 16 L is taken from the first vial and transferred into a fourth vial, which contains 184 L of PBS (total volume of 200 L). The fourth vial is gently agitated for 1 minute to homogenize the solution. With a micropipette, 4 L is taken from the first vial and transferred into a fifth vial, which contains 196 L of PBS (total volume of 200 L). The fifth vial is gently agitated for 1 minute to homogenize the solution. With a micropipette, 1.2 L is taken from the first vial and transferred into a sixth vial, which contains 298.8 L of PBS (total volume of 300 L). The sixth vial is gently agitated for 1 minute to homogenize the solution.
(75) Once the six solutions are prepared (1.sup.th to 6.sup.th vials), 20 L of each sample is transferred into 180 L of cells in a 96-well plate (total volume of 200 L). Each sample is measured in triplicate. The final concentrations of the first, second, third, fourth, fifth and sixth samples will be 250 M, 100 M, 40 M, 20 M, 5 M and 1 M, respectively.
(76) Procedure for the Encapsulation Process
(77) One important aspect of the invention is the capacity of the SHS and f-SHS to encapsulate a wide variety of guest molecules (e.g., drugs, proteins, DNA) due to its surface and internal porosity. Thus, the present invention provides three encapsulation protocols as illustrated in
(78) Procedure for Osmotic Gradient Method where a Guest is Incubated with a Previously Made (ii) SHS
(79) Once the SHS (5 mM of ImAGpD2OH, 2M KI) is formed at 40 C. (also a temperature equal or above the T.sub.cp) and neutral pH, the amount of desired guest dissolved in PBS is added to the colloidal suspension. For example, to add 100 equivalents of Doxorubicin, 100 L of Dox is added with a micropipette to the SHS colloidal suspension. The mixture is stirred for 1-3 min at 40 C., and then the fixing process procedure described before is carried out to obtain 1.65 mL of f-SHS (0.303 mM of ImAGpD2OH).
(80) Procedure for Osmotic Gradient Method where a Guest is Incubated with a Previously Made (iii) f-SHS
(81) Once the f-SHS solution is prepared at neutral pH as previously described, 200 L of f-SHS (0.303 mM of ImAGpD2OH) is transferred in an Eppendorf tube followed by the addition in equivalents of the guest that will be encapsulated in f-SHS. For example, for 5 equivalents of each dextran Texas Red (DTR) polymer solution 37 L of 0.0685 mM is added to the corresponding Eppendorf tube. Then all samples are incubated for 1 h at 35 C. with movement.
(82) Procedure for Delivery Studies
(83) Induced pH Disassembly Guest Release
(84) As shown in
(85) The isothermal disassembly process can be observed once the cloudy colloidal suspension becomes completely soluble after acid titration from pH 7.2 to pH 5.7. But the disassembly process can also be followed with .sup.1H NMR by comparing the NH region where the characteristic NH peak of neutral nanoglobular octameric oSGQ (which is the component of microglobular SHS) appears, with the characteristic peaks of the resulting water-soluble nanoglobular hexadecameric hSGQ. This isothermal pH-responsive behavior is a convenient method to release encapsulated cargo for delivery applications.
(86) Biorelevant pH- and thermo-responsive properties of the resulting SGQ formed by ImAGpD2OH (or ImAGhD2OH) makes this derivative an excellent means for encapsulation applications. Encapsulation of the anticancer drug doxorubicin (DOX) by thermoresponsive SGQ that self-assembled at 32 C. into microglobules was previously reported. Now, the present invention provides encapsulation studies of DOX with oSGQ by pH-induced encapsulation as shown in
(87) Slow Controlled Release
(88) In addition to the ability of f-SHS to encapsulate different types of guests and their potential use as imaging probes, the guest-release properties are also explained. The guest leakage of non-encapsulated guest for the samples was evaluated by measuring the aqueous phase by UV-Vis spectroscopy. However, the amount of non-encapsulated guest leakage in the aqueous phase was below the detection limit of the instrument (less than 1 M). For that reason, the present invention was focused in the DOX release with time by increasing the equivalents of encapsulated DOX in the f-SHS.
(89) Six different 150 L solutions of 5 mM SHS were prepared and placed in a water bath at 40 C. To each of these solutions, different equivalents of DOX were loaded to obtain solutions with 60, 80, 100, 200, 300, and 400 equivalents of DOX. It should be noted that to prepare these samples, different volumes of a 487 M solution of DOX were added, so that the resulting concentration of SHS was not the same in each of the solutions. However, since the same initial volume of SHS was used, the amount of moles in solution is constant. To examine the encapsulation capacity of the microglobules, the solutions were fixed by adding them dropwise to a vial containing 1 mL of 1PBS buffer to form two phases. An aliquot was obtained from the aqueous phase to determine the amount of non-encapsulated DOX in solution by UV-Vis as shown in
(90) In contrast to other delivery vehicles that release their guest prematurely, f-SHS showed a slow-release behavior that is very convenient for drug-delivery applications. According with the performed UV-Vis studies, f-SHS has great affinity for DOX but the attractive feature is that the encapsulated cargo can be released to the aqueous phase outside the colloidal phase of f-SHS. For in vivo applications, it is necessary that this kind of guest reaches the target tissue without affecting other surrounding tissues. Many NPs are limited to covalent chemistry to retain their encapsulated guest, but with f-SHS a system is provided that can survive higher dilutions without losing the non-covalent encapsulated cargo.
(91) For the time-release experiments, 100 L of SHS colloid were added to a 7 mL scintillation vial at 40 C. containing 1 mL of PBS at pH 7.2 or sodium acetate buffer at pH 5. For both pH 5 and 7.2, six samples were prepared containing the two phases (colloidal and aqueous non-colloidal) to measure the concentration of non-encapsulated and released DOX in the aqueous (non-colloidal) phase from the f-SHS colloidal phase. 100 L of the aqueous phase were extracted at different time points (5 min, 15 min, 30 min, 60 min, 146 min and 24 h) to be analyzed by UV-Vis as shown by
(92) The f-SHS are Biocompatible (Non-Cytotoxic) Particles that are Taken Up by Neuroblastoma Cancer Cells (SH-SY5Y) In Vitro, which Make them Suitable as Probes for Biological Studies
(93) We discovered that f-SHS could be internalized in neuroblastoma cells (SH-SY5Y; the most common extracranial solid cancer in children), enabling the development of new biological probes as illustrated in
(94) Encapsulation of dextran labeled with Texas Red of 3 kDa (DTR-3) into f-SHS is a convenient strategy to prepare fluorescent biological probes because it does not require further covalent modifications to keep the fluorescent guest encapsulated inside the f-SHS. For example, a 24 h incubation study of SH-SY5Y cells with f-SHS@DTR-3 demonstrated that the cellular uptake of these cells was reproducible as shown in
(95) Cell Culture Method
(96) SH-SY5Y and HEK-293 cell lines were purchased at ATCC. SH-SY5Y cells are a thrice-cloned line obtained from a metastatic bone marrow tumor of a human female. DAPI nuclear stain used was the kit VECTASHIELD mounting medium from VECTOR laboratories. In the case of Hoechst 33342 (Hoechst) nuclear stain; a kit called NucBlue Live Ready Probes Reagent from Life Technologies, Inc. was used (Lysotracker kit was also obtained from Life Technologies, Inc.). The rest of the reagents for the cell culture treatments were purchased from Aldrich and used without further treatment.
(97) Both cell lines were cultured in Dulbecco's Eagle's Medium/Nutrient Mixture F-12 (DMEM F-12), that was supplemented with 10% fetal bovine serum (FBS) (from Aldrich), 100 units/mL of penicillin, 100 g/mL of streptomycin and 0.25 g/mL of amphotericin B. Cells were incubated in humidified air containing 5.0% CO.sub.2 at 38.0 C. Cells were placed in wells at 110.sup.6 per well in a 48 well plate (1.0 mL). The cell media (pH=7.4) were the cells were cultured was changed the next day, and every two days after that.
(98) b) Fluorescence Confocal Microscopy
(99) The confocal microscopy images were performed in a Confocal Zeiss LSM 510 META on an Axiovision Z1 microscope with an excitation range of (514-515) nm and emission range of (565-615) nm from the Confocal Microscopy Facility at the University of Puerto Rico (CIF-UPR). The microscope objectives used to obtain the confocal images were Plan-Apochromat 63/1.40 Oil DIC M27 and EC Plan-Neo Fluar 40/0.75. Laser wavelength excitation wavelengths were 561 nm (for f-SHS, DOX and DTR 3 kDa), 405 nm (for DAPI and Hoechst) and 633 nm (for Lysotracker). The emission filters used are 588-738 for ChS1, BP 420-480 for Ch2 and BP 575-615 IR for Ch3. ChS1 of 652-738 was used for Lysotracker and Ch3 of LP 575 was used for incubation experiments of 8 days. All the fluorescence confocal microscopy images, z-stacks and movies were processed in Zeiss LSM Image Browser software
(100) The f-SHS Particles are a Viable Drug Delivery System as Demonstrated by the (In Vitro) Delivery, and Controlled Release, of the Anticancer Drug DOX into Neuroblastoma Cancer Cells.
(101) The evidence from the CLSM experiments shown in
(102) The MTT assay confirmed the non-toxicity (for the concentration range tested) of the f-SHS, in contrast to other reported systems that need modifications to reduce toxicity. The f-SHS@DOX particles loaded with relatively large amounts of DOX show lower toxicity, in the short term relative to similar concentrations of DOX alone (e.g., 0.2 M DOX is cytotoxic; f-SHS@DOX with 39.0 M DOX is less cytotoxic; f-SHS@DOX with 246 M DOX kills 50% of the cancer cells after 24 h). Due to the controlled sustained release, the invention has the same cytotoxic effect in the long run as shown in
(103) MTT Assay Protocol
(104) These experiments were performed using an MTT Cell Proliferation Assay purchased from ATCC. First, cells were placed in wells at 110.sup.6 per well in a 96 well plate (100 L). They were left there for 24 h until cells adhered to the plate. When 80%-90% of confluence was reached, the cells were treated at different time points with the performed treatments including the positive control of 300 M H.sub.2O.sub.2. After treatment, 100 L of fresh medium per well were added, followed by 10 L of MTT reagents. Cells were then incubated for 8 h or overnight with the reagent until a precipitate was visible. Next, 100 L of detergent reagent were added to the sample, without removing any of the medium. Then the treated cells were left at 25 C. in the dark for 4 h to measure the absorbance at 570 nm excitation wavelength.
(105) The f-SHS as a Viable Transfection Agent, Suitable for Gene-Delivery Applications
(106) Another attractive feature of the f-SHS particles is their potential as a versatile platform technology for delivering nucleic acid therapeutics. As described earlier the f-SHS particles can encapsulate short DNA oligonucleotides (
(107) Considering the significant difference in size between DNA oligonucleotides and pDNA, we needed to confirm the interactions between the latter and f-SHS particles. Incubation of f-SHS particles and two different pDNAs encoding for green fluorescent protein (pGFP) and crimson fluorescent protein (pCri), this was followed by drop-casting (depositing a drop on a surface and letting it air dry at 36 C.). Scanning electron microscopy (SEM) images of the samples reveal a dramatic morphological transformation as shown in
(108) CLSM measurements confirm that both, f-SHS@pCri and f-SHS@pGFP particles are also taken up by neuroblastoma (SH-SY5Y) cells.
(109) The distribution of the expressed protein is different in experiments with LA2000, f-SHS@pGFP and f-SHS@pCri as shown in rows (b) of
(110) Procedure to Perform Plasmid Transfection Studies with f-SHS
(111) A volume of 300 L of each f-SHS@plasmid was added to corresponding wells. In the case of LIPOFECTAMINE sample, only 100 L of each preparation were added to each well. For each individual plasmid control sample, these volumes were added to each well: 20 L of p-E2Crimson and 30 L of p-GFP. All cells samples were incubated for 15 h at 37 C. and 5% CO.sub.2 without movement as a first checkpoint of behavior. Then, the incubation of cells was continued at 37 C. for 3 and 8 days. After each checkpoint, confocal microscopy images were taken to visualize or analyze the current encapsulation/intake/protein expression effect. Two additional samples were prepared to be evaluated at Day 3 checkpoint: 0.164 mM ImAG at pH 6.1 encapsulating 10 L of p-GFP; and 0.164 mM ImAG at pH 6.1 encapsulating 15 L of p-E2Crimson. Samples were prepared individually for the three different checkpoints to avoid contamination during their management and transport to the confocal microscopy facilities. (Confocal Zeiss LSM 510 META on an Axiovision Z1 microscope from the Confocal Microscopy Facility at the University of Puerto Rico (CIF-UPR)).
(112) While the embodiments of the present invention have been showed as described, various alterations can be made without deviating from intended utility; and all such modifications are intended to be covered within this patent.