This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.
We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 ± 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.
In recent years, there has been a growing interest in the engineering of nanoparticles for such biomedical applications as drug delivery and bioimaging1,2,3,4. The multifunctionality of nanoparticle-based systems often necessitates incorporating multiple components within one formulation. The building blocks that are based on lipids or polymers often differ in terms of their physicochemical properties as well as their biocompatibility and biodegradability, which ultimately might affect the function of the nanostructure1,5,6. Biologically derived materials, such as proteins and peptides, have long been recognized as promising components of multifunctional nanostructures due to their sequence flexibility7,8. Peptides self-assemble into highly ordered supramolecular architectures forming helical ribbons9,10, fibrous scaffolds11,12, and many more, thus paving the way to building biomolecule-based hybrid nanostructures using a bottom-up approach13.
Peptides have been explored for applications in medicine and biotechnology, especially for anticancer therapy14 and cardiovascular diseases15 as well as for antibiotic development16,17, metabolic disorders18, and infections19. There are over a hundred of small-peptide therapeutics undergoing clinical trials20. Peptides are easy to modify and fast to synthesize at low cost. In addition, they are biodegradable, which greatly facilitates their biological and pharmaceutical applications21,22. The use of peptides as structural components includes the engineering of responsive, peptide-based nanoparticles and hydrogel depots for controlled release23,24,25,26,27, peptide-based biosensors28,29,30,31, or bio-electronic devices32,33,34. Importantly, even short peptides with two or three amino-acid residues that include phenylalanine were found to guide the self-assembly processes35,36,37 and create stabilized emulsions38.
Platinum-based drugs, owing to their high efficacy, are used in many cancer treatment regimens, both alone and in combination with other agents39,40. Platinum compounds induce DNA damage by forming monoadducts and intrastrand or interstrand cross-links. The Pt-DNA lesions are recognized by the cellular machinery and, if not repaired, lead to cellular apoptosis. The most important mechanism, by which Pt(II) contributes to cancer cell death, is the inhibition of DNA transcription41,42. However, the benefits of platinum therapy are diminished by systemic toxicity of Pt(II) that triggers severe side effects. This leads to lower clinical dosing of Pt(II)43, which often results in sub-therapeutic concentrations of platinum reaching the DNA. As a consequence, the DNA repair that follows contributes to cancer cell survival and acquiring Pt(II) resistance. The platinum chemo-resistance is a major problem in anticancer therapy and the main cause of treatment failure44,45.
We have developed a stable nanosystem that encapsulates the Pt(II) agent in order to provide a shielding effect in systemic circulation and to diminish the Pt(II)-induced side effects. The system is based on an oleic acids-Pt(II) core stabilized with a KYF tripeptide to form a nanoemulsion (KYF-Pt-NE)46. The building blocks of KYF-Pt-NE, the amino acids of the tripeptide as well as the oleic acid, have the Generally Recognized As Safe (GRAS) status with Food and Drug Administration (FDA). The KYF-Pt-NE is prepared by using a nanoprecipitation method47. In short, the oleic acids-Pt(II) conjugate is dissolved in an organic solvent and then added dropwise to an aqueous KYF solution (Figure 1) at 37 °C. The solution is stirred for several hours to allow self-assembly of the KYF-Pt-NE. The nanoemulsion is concentrated in 10 kDa centrifugal concentrators and washed three times with water. The chemical modification of the KYF with a fluorophore allows the synthesis of fluorescent FITC-KYF-Pt-NE suitable for biomedical imaging.
1. Synthesis of the Oleic Acids–Platinum(II) Conjugate
2. Synthesis of KYF-Pt-NE, and the FITC-labeled Nanoemulsion FITC-KYF-Pt-NE
3. Confocal Imaging of the Cellular Uptake of FITC-KYF-Pt-NE
4. Drug Release Studies
5. Cell Culture Methods
Representative TEM image of KYF-Pt-NE prepared using this protocol is shown in Figure 2A. The KYF-Pt-NEs are spherical in morphology, well dispersed, and uniform in size. The core diameter of the KYF-Pt-NEs, measured directly from three TEM images with a minimum of 200 measurements done, is 107 ± 27 nm. The hydrodynamic diameter of KYF-Pt-NE, analyzed using dynamic light spectroscopy (DLS), was found to be 240 nm with a polydispersity index of 0.156. The Zeta potential of KYF-Pt-NE in water was determined for three independent syntheses with the average value of -60.1 mV. The high magnitude of the potential indicates good colloidal stability of the formulation, and the negative surface charge is attributed to ionized COO– surface groups of oleic acids. The isoelectric point of KYF is 8.59, and therefore it is positively charged at neutral pH.
The ability of the nanoemulsions to cross the cellular membrane was examined using the fluorescently labeled FITC-KYF-Pt-NE and ovarian cancer cell lines. The results are presented in Figure 2B. It can be clearly seen that the FITC-KYF-Pt-NEs (green) are distributed within the cytosol but were not yet associated with lysosomes (red). This result demonstrates that KYF-Pt-NEs enter cells and can serve as intracellular drug delivery vehicle.
The stability of KYF-Pt-NEs and FITC-KYF-Pt-NEs was assessed with DLS. The diameter of the nanoemulsions in water was measured over several months and the results are presented in Figure 3. The hydrodynamic diameter of both formulations slowly increased during 4 months in storage, to 320 nm (KYF-Pt-NE) and 240 nm (FITC-KYF-Pt-NE), but the nanoscale dimensions were preserved. This result suggests that the KYF tripeptide effectively stabilizes nanoemulsions over long periods of time.
The Pt(II) encapsulation efficiency and release from the nanoemulsion were measured with AAS. The Pt(II) concentration in KYF-Pt-NE was established to be 10 wt.%. The Pt(II) release from KYF-Pt-NE at pH 7.4 (physiological), 6.8 (tumor's interstitium)49, and 5.0 (endosomal)50 is presented in Figure 4A. The Pt(II) release is the slowest at pH 7.4 with only 20.8% of Pt(II) released after 4 h, while at pH 6.8 and 5.0 the release was 32.8% of and 47.5%, respectively. The same trend continued after 24 h and after 6 days. This result indicates that Pt(II) release from KYF-Pt-NE is pH dependent, and it can be delayed in the systemic circulation, and accelerated once the nanoemulsions translocate into tumor.
The biological activity of the KYF-Pt-NE was established in vitro using the same ovarian cancer cell lines as in the imaging studies. The cells were incubated with KYF-Pt-NE for 72 h, at KYF-Pt-NE concentrations corresponding to IC50 for each cell line. The viability was assessed using the MTT assay and the results were compared to cells only, KYF-NE, oleic acids-Pt(II) conjugate, carboplatin, and cisplatin. The results of biological activity of KYF-Pt-NE are shown in Figure 4B. The KYF-Pt-NE reduced the viability of isogenic cell lines A2780 (Pt sensitive) and CP70 (Pt resistant) by 44.3% and 46.2% respectively. Carboplatin, the clinically relevant analogue, decreased the viability by 18.5% (A2780) and 9.6% (CP70) only. The same trend of greater KYF-Pt-NE effect on cell death was observed across other cell lines as well. The viability of Pt(II) sensitive TOV-21G cells was reduced by 55.9% after incubation with KYF-Pt-NE, while carboplatin resulted in lowering the viability by just 16.5%. In OV-90 cells with intermediate Pt(II) resistance, the viability was lowered by 55.3% (KYF-Pt-NE) and 23.9% (carboplatin). The two resistant cancer cell lines, ES-2 and SKOV-3, showed reduction in viability by 45.9% and 54.3%, respectively, for KYF-Pt-NE, and 10.3% (ES-2) and 16.8% (SKOV-3) for carboplatin.
The biological activity of KYF-Pt-NE versus cisplatin was also compared. Cisplatin is the Pt(II)-based agent of first generation that is no longer favored in the clinic due to its toxicity profile51. In two cell lines, SKOV-3 and TOV-21G, the viability reduction was greater by 15% and 40%, respectively, for KYF-Pt-NE than for cisplatin. In the remaining cell lines, the activity of KYF-Pt-NE was comparable to cisplatin (A2780, CP70, OV-90), or slightly lower (ES-2). The oleic acids-Pt(II) conjugate was also found to be biologically active. However, KYF-Pt-NE showed higher reduction in viability than the conjugate in majority of the cell lines tested, indicating the significance of nanoformulation in Pt(II) activity.
The biological applications of nanoparticulate systems require stability in biologically relevant media, thus the stability of KYF-Pt-NE was evaluated in 20% fetal bovine serum (FBS) and the results are presented in Figure 4C. We detected no evidence of KYF-Pt-NE opsonization in serum after one day of incubation.
Figure 1. The components of the nanoemulsions (top) and schematic of the nanoemulsion preparation (bottom). Please click here to view a larger version of this figure.
Figure 2. TEM image of KYF-Pt-NE (A) and confocal microscope images of FITC-KYF-Pt-NE (green) uptake by ovarian cancer cells (nuclei are blue and lysosomes are red) (B). Scale bars are 10 μm. This Figure has been adapted with permission from Bioconjugate Chemistry 2018, 29, 2514-2519.46 Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
Figure 3. Stability of KYF-Pt-NE and FITC-KYF-Pt-NE in water. Each point represents the mean and standard deviation of N=3 and p <0.005. This Figure has been adapted with permission from Bioconjugate Chemistry 2018, 29, 2514-2519.46 Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
Figure 4. KYF-Pt-NE stability and biological activity. (A) Pt(II) release from KYF-Pt-NE in PBS buffer at different pHs (PBS, 37 °C). Cellular viability of different ovarian cancer cell lines after 72 h incubation with KYF-Pt-NE (B). Each column represents the mean and standard deviation of N=3 and p <0.005. The concentrations are constant in each cell line and are as follows: 4.92 μM (A2780), 8.80 μM (CP70), 2.46 μM (TOV-21G), 9.84 μM (SKOV3), 7.38 μM (ES-2), 19.7 μM (OV-90). The concentration of KYF-Pt-NE and oleic acids–Pt(II) conjugate was adjusted with respect to Pt(II) content measured by AAS. Abbreviations: “Carbpt” – carboplatin; “KYF-NE” – KYF tripeptide-coated nanoemulsion; “KYF-Pt-NE” – KYF tripeptide-coated nanoemulsion containing Pt(II); Cispt – cisplatin; Pt-oleic – oleic acids–Pt(II) conjugate. (C) KYF-Pt-NE stability in serum. This Figure has been adapted with permission from Bioconjugate Chemistry 2018, 29, 2514-2519.46 Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
Critical steps in the nanoemulsion synthesis include adjusting the molar ratio of the substrates, maintaining temperature and flow rate control during oleic acids–Pt(II) addition, providing sufficient time for self-assembly, and purifying the product using a centrifugal concentrator column. These parameters influence the size and morphology of the KYF-Pt-NE; thus, it is particularly important to maintain the proper molar ratio and adjust the synthetic conditions correctly.
The ratio of the substrates during the nanoemulsion synthesis (step 3) is crucial for the self-assembly process and determines the final size of the product. The KYF to oleic acid-Pt(II) molar ratio is 1:3, and the optimal concentration of the KYF tripepide in water is 0.2 mM. In addition, the organic solvent used to dissolve the oleic acid-Pt(II) conjugate in step 3.1 has to be miscible with water, compatible with the filtration system, and evaporative easily at room temperature.
One of the critical steps is the dropwise addition of the oleic acid-Pt(II) conjugate to the KYF solution (step 3.4). The flow should not be slower than 0.1 mL/min and not faster than 0.2 mL/min, because a sub-optimal speed can induce the precipitation of the nanoemulsion. Also, the oleic acids–Pt(II) conjugate should be added to the KYF solution at 37 °C while stirring the mixture on a stir plate at 600 rpm. Once all of the oleic acid-Pt(II) has been added, the solution should be kept at room temperature and the stirring speed reduced to 150 rpm. Step 3.5 should be carried out at room temperature. The optimal time for the self-assembly of the KYF-Pt-NE (step 3.5) is 24 hours.
There is a risk that the product may precipitate while the nanoemulsion is being pre-concentrated. Therefore, it is recommended that the nanoemulsion be diluted with additional water before being centrifuged in a centrifugal concentrator and spun no faster than 2,465 x g. The diluted nanoemulsions should be added to the centrifugal concentrator at aliquots between spins, and the nanoemulsion should be mixed in the filter before the next portion is added.
The ability to form nanoemulsions with fatty acids derivatives other than oleic acid has not been tested and is yet to be determined. Future applications may include the use of different peptides to facilitate the co-assembly with oleic acid. Also, oleic acid conjugates with non-platinum-based drugs may be used as potential cores of nanoemulsions.
The authors have nothing to disclose.
We gratefully acknowledge financial support from the National Cancer Institute, grant SC2CA206194. No competing financial interests are declared.
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) |
ANASPEC INC.: | AS-20376 | SPPS |
4-well chamber confocal dish | Lab-Tek II, Thermo Fisher Scientific | 154526 | For imaging |
6-bromohexanoic acid | Chem-Impex INT’L INC. | 24477 | Click modification for peptide |
A2780 | Generously doanted by professor John Martignetti from The Mount Sinai Hospital | Ovarian cancer cell line | |
Barnstead Nanopure | Thermo Fisher | D11901 | water filtration system |
BUCHI rotavapor R-3 | Buchi | Z568090 | For solvent removal and sample drying |
Centrifuge 5810 R | eppendorf | 5811F | For platinum complex separation |
Cis-dichlorodiamineplatinum (II) 99% | Acros Organics | 19376-0050 | in vitro tests |
CP70 | Generously doanted by professor John Martignetti from The Mount Sinai Hospital | Ovarian cancer cell line | |
Digital water bath | VWR | 97025-134 | For warming up media for cell culture |
Dynamic Light Scattering (DLS) | Brookhaven Instrument Corporation | For nanoparticle size measurments | |
ES-2 | ATCC | CRL-1978 | ovarian cancer cell line |
Fmoc-L-Lys(Boc)-OH 99.79% | Chem-Impex INT’L INC. | 00493 | SPPS |
Fmoc-L-Phe 4-alkoxybenzyl alcohol resin (0.382 meq/g), | Chem-Impex INT’L INC. | 01914 | SPPS |
Fmoc-LTyr(tBu)-OH 98% | Alfa Aesar | H59730 | SPPS |
HERACELL 150i CO2 incubator | Thermo Scientific Fisher | incubator | |
High pressure syringe pump | New Era | 1010-US | For platinum complex addition in nanoparticle synthesis |
Hotplate/stirrer | VWR | 12365-382 | For sample stirring and heating |
LAMP-1 Antibody(cojugated with Alexa Fluor 647) | Santa Cruz Biotechnology | sc-18821 AF647 | For imaging |
N,N-diisopropylethylamine (DIPEA) | Oakwood Chemical | 005027 | SPPS |
Ninhydrin 99% | Alfa Aesar | A10409 | Kaiser test |
Oleic acid | Chem-Impex INT’L INC. | 01421 | For platinum complex synthesis |
OV90 | ATCC | CRL-11732 | Ovarian cancer cell line |
PBS | Corning | 21-031-CV | For cell wash |
Permount mounting medium | Fisher Chemical | SP15-100 | For imaging |
Phenol | Fisher Chemical | A92500 | Kaiser test |
Phosphotungstic acid | Fisher Chemical | A248-25 | negative stain for TEM |
Piperidine 99% | BTC | 219260-2.5L | SPPS |
Platinum AAS standard soultion | Alfa Aesar | 88086 | 1000ug/ml for calibration curve |
Propargyl bromide 97% | Alfa Aesar | L10595 | For alkyne modification of fluoresceine |
Scientific biological cabinet | Thermo Scientific Fisher | 1385 | Bio-hood for cell culture |
Self-Cleaning Vacuum System | Welch | 2028 | Vacuum pump for rotavapor |
Silver nitrate | Acros Organics | 19768-0250 | Cisplatin activation |
SKOV3 | ATCC | HTB-77 | Ovarian cancer cell line |
Sodium hydroxide | Fisher Scientific | S313-1 | For platinum complex synthesis |
Tin (II) chloride | Sigma Aldrich | 208256 | Test for Platinum presence |
TOV21G | ATCC | CRL-11730 | Ovarian cancer cell line |
Trifluoroacetic acid 99% (TFA) | Alfa Aesar | L06374 | SPPS |
Triisopropylsilane (TIPS) | Chem-Impex INT’L INC. | 01966 | SPPS |
Triton-X | Sigma Aldrich | T8787-100ML | For imaging |
Uranine powder 40% | Fisher Scientific | S25328A | For alkyne modification of fluoresceine |
Vivaspin 20 (10000 MWCO) | Sartorious | VS2001 | For Nanoparticle wash and condensation |
VWR Inverted Microscope | VWR | 89404-462 | For cell culture monitoring |