Inorganic/Organic Silica Polyfluorene Hybrid Fluorescent Nanoparticles: Surface Modifications and Layer-by-Layer depositions of CPNPs
Author: Camilo Hernandez
Research Professor: Dr. Joong Ho Moon
Surface modifications of polyfluorene organic inorganic composite nanoparticles have been studied for incorporation of 3-aminopropyltrimethoxysilane. The highly fluorescent emission given off by polyflorene polymers makes them great candidates for bio imaging of cellular uptake and drug delivery. By attempting to incorporate an amino group into the inorganic silica surface matrix of a polyfluorene hybrid nanoparticle, a positive surface charge may be attained, which will be useful in trafficking negatively charged DNA through ionic interactions. The experimental results show that an increase in amino groups can be achieved by carefully controlling the addition of alkoxysilane monomer, tetramethylorthosilicate, and the phase transfer catalyst tetramethylammonium hydroxide. Also considers, is using layer-by-layer deposition as a tool for coating the surface of the nanoparticles with positively charged polymers, as an alternative to the amino group surface modification of the inorganic matrix.
Highly fluorescent polymers, in the recent years, have been used as a tool for biomedical applications, such as bioimaging, biosensing, precise timed release of biological molecules, and in tissue engineering. Conjugated Polymer have exceptional photoluminescence quantum yields (PLQYs), making them great candidates for the hydrophobic carbon backbone nanoparticles. Conjugated polymer nanoparticles (CPNPs) can be aggregated from polymers by two methods: 1) Microemulsion; dispersion made of water, oil, and surfactant(s) that makes an isotropic and thermodynamically stable system with dispersed domain diameter varying approximately from 1 to 100 nm, usually 10
to 50 nm, or 2) Nanoprecipitation; a technique used in which the precipitation of a material is controlled by aggregating the material after rapid injection to a non-solvent (usually water) that is miscible with the polymer solvent . In this experiment the second method will be explored, because of its ability to make smaller (10-200 nm) and more monodispersed CPNPs.
Polyfluorenes are a group of conjugated polymers with a cyclic 9-carbon backbone including 2 benzene rings. As shown bonds on position 2 and 7 usually connect the monomers, while side chains are connected on position 9 (Fig. 1). Hybrid inorganic organic polyfluorene polymers can be synthesized by coupling the polymers with an alkane side chain containing an inorganic pedant charged group such as silica. These pedant polymerizable charged groups can provide ionic or covalent surface modification between the CPNP and the inorganic host matrix. By using the procedure provided by Dr. Behrendt in J. Mater. Chem. C, 2013, 1, 3297, and using the polyfluorene conjugated polymer named in the paper as P5 (figure 2). P5 is, reported by GPC, on having a molecular weight of 21,000 9. Hybrid inorganic–organic composite nanoparticles were formed through nanoprecipitation of this polymer into an aqueous solution of tetramethylammonium hydroxide (TMAOH). TMAOH is used as a phase transfer catalyst and a surfactant in the synthesis of the ferrofluids, which inhibit nanoparticle aggregation, and are especially helpful in the presence of the tetraalkoxysilane monomer, tetramethylorthosilicate (TMOS).
Resulting nanoparticles from the published paper were measured to have diameter/zeta potentials of 136 nm/-46.3 mV (no TMOS), 139 nm/-39.5mV (1:1 polymer to TMOS ratio by weight), 167 nm/ -51.7 mV (1:5 polymer to TMOS ratio by weight), and 214nm/ -55.5mV (1:10 polymer to TMOS ratio by weight) 6. As expected nanoparticle radius increases and surface zeta potential decreases with higher concentrations of TMOS used, and at no TMOS concentrations the resulting radius is 68nm providing the absolute minimum particle radius. Also, fluorescence is expected to increase with rising concentrations of nanoparticle side chain cross-linking with the TMOS matrix due to high-energy -conformation.
Surface modifications will be explored by crosslinking 3-aminopropyltrimethoxysilane (3-APTMS) with the inorganic silica side chains and the TMOS matrix. Due to the positive charge on the amino groups at pH 7, the expected results of successive cross-links will be an increase in surface zeta potential. Due to disruption of the high-energy -conformation, fluorescence is expected to decrease. Layer-by-layer deposition, a cheap and simple method for forming alternating layers of oppositely charged materials offers a method for making ionic modifications to the resulting organic/ inorganic matrix hybrid CPNPs. With microinjections of nanoparticle solution into solutions of alternatingly oppositely charged nanoparticle solutions or solutions of biological material to be trafficked, and subsequent washing steps in between, the CPNPs radius may be controlled with precision to the nm range12.
Experimental Materials and Methods:
UV-visible absorption spectra were recorded using a Varian Carry 50 Bio UV-visible spectrophotometer, in either THF (linear polymers) or water (nanoparticles) at room temperature (25 oC). Fluorescence spectra and PLQY was recorded and calculated using a Horiba Jobin Yvon Fluorolog Model FL-1039/40, in either THF (linear polymers) or water (nanoparticles) at room temperature (25 oC). The size and Zeta potentials of the nanoparticles were measured using a Malvern Zetasizer Nano S, at room temperature, in 18 deionized water. For the zeta measurements 700 of the sample was used per measurement for a total of 6 measurements, and for size measurements 3 measurements of 100 mL were used.
Nanoparticle Formation of P5:
First, making a 5000-ppm polymer solution consisting of 10 mg of P5 in 10 g of THF (step 1)*. After dissolving the polymer by stirring for 48 hours in the dark, excess insoluble matter was filtered out using a 0.45 nm PTFV filter resulting in a solution of unknown decreased concentration (step 2). To 750 L of this polymer solution, 75 L of pure (6.72M) TMOS was added with stirring for 5 minutes (step 3). Then, the polymer solution was rapidly injected into 3 mL of 24 mM TMAOH in 18 DI water with stirring for 5 minutes (step 4). The resulting nanoparticle solution was transferred to dialysis chamber (2 kDa MWCO) and flushed with 1 L of water (step 5). Resulting purified nanoparticle solutions were filtered using P8 filter paper and stored in lightproof vials (step 6). Procedures, for CPNPs formation under diluted concentrations of TMAOH used 24 TMAOH in step 4. Procedures following 3-APTMS surface modifications added 3.75 L of 5.2M, 0.52M, 0.052M or 0.0052M 3-APTMS during step 3.
Results and Discussion:
A total of 12 CPNPs were synthesized with varying concentrations of TMOS, 3-APTMS and TMAOH, testing the difference aggregation, absorbance, fluorescence PLQYs, and average particle size and zeta potentials. The full lists of results are posted on table 1 with conditions that differ from the published procedures. NP1 was synthesized following the published procedures (24 mM TMAOH) and resulted with a quantum yield of 37.8%. NP2 was synthesized using 24 TMAOH and resulted with a PLQY of 29.8%. This is indicative that there is less crosslinking of the inorganic matrix with the nanoparticles. During both these formations there was minimal precipitation. Cellular studies of NP2 revealed high fluorescence but very low cellular uptake. Nanoparticle size measured resulted in a radius of 84.59 nm and a zeta potential of -37.5 mV (fig. 3 and 4). Under these conditions the average particle radius increased by 16.59 nm, and with each silica layer adding about 1 nm to the radius, it is estimated that 16 layers of silica were incorporated. The absorbance and fluorescence spectra are below in figure 5. When compared to using 24 mM TMAOH, using diluted concentration of the phase transfer catalyst decreases CPNP radius, evidence that less of the TMOS inorganic matrix has layered onto the CPNP surface. Therefore, using 24 mM we can predict that the more than 16 layers of silica have been deposited. NP3 was synthesized using 3.75 μL of 5.2 M 3-APTMS and vigorous precipitation and gelation occurred resulting in a gel like solutions, and NP4 was synthesized using 3.75 μL of 5.2M 3-APTMS and 24 μM TMAOH, precipitation and gelation still occurring although not as intensely as the previous trial. These results are indicative that the using larger concentrations of TMAOH will promote crosslinking of the 3-APTMS, inorganic matrix and nanoparticles, at the expense of aggregation, precipitation and gelation.
The three following CPNPs formed used 24 mM TMAOH with varying concentrations of 3-APTMS. NP5 used of 0.52M 3-APTMS and resulted in excess precipitation. NP6 used of 0.052M 3-APTMS and NP7 used of 0.0052M 3-APTMS and although there was minor precipitation for both, the PLQY resulted as 17.3% and 3.33% respectively. Next, the following two nanoparticles formed used a constant 3.75 μL 0.052M 3-APTMS with varying concentrations of TMOS. NP8 used 7.5 μL pure TMOS, resulting in almost no precipitation, and NP9 used 750 μL pure TMOS resulting in vigorous gelation. These results show that TMOS concentration also plays an important part in nanoparticle aggregation.
The next three trials varied TMOS concentrations to allow for successful incorporation of 3-APTMS into the crosslinks without precipitation. NP10 used 75 μL of 0.0672 M TMOS and 3.75 μL of 5.2 M 3-APTMS resulting in major precipitation. NP11 used 75 μL of 0.00672 M TMOS and 3.75 μL of 5.2 M 3-APTMS resulting with minor precipitation and a PLQY of 6.86%. NPs used 75 μL of 0.000672 M TMOS and 3.75 μL of 5.2 M 3-APTMS resulting with minor precipitation and a PLQY of 2.49%. Decreased PLQYs are evidence that a higher concentration of 3-APTMS has been adopted into the nanoparticle matrix crosslinks.
The average surface area of the resulting particles (without TMOS) is calculated to be 58,000 nm2. Based on the molar mass and density the calculated area of one molecule of TMOS equals nm2. The ratio of TMOS per nanoparticle results in 61,000 TMOS molecules per nanoparticle for saturation with a single layer. If reacting 750 of 2.12 M P5 CPNP solution it will theoretically take 144 6.72M TMOS to do this.
As stated in the introduction PLQY are expected to decrease with an increase concentration of incorporated 3-APTMS. Based on the results, although the concentrations of 3-APTMS were decreased in NP7, the PLQYs recorded show the second lowest value, evident of higher amino incorporation. The decrease in [3-APTMS] in the presence of high [TMOS] or vice versa past a critical value results in excess precipitation. Consequently, NP7 and NP12 showed more aggregation than normal resulting in absorbance decreased by a factor of 10. NP11 shows to have incorporated the highest amount of 3-APTMS into inorganic crosslinks of the TMOS matrix with minimal amount of precipitation. NP6, which used higher concentrations of TMOS and 3-APTMS shows to have incorporated around half the 3-APTMS that the conditions of NP11 with minimal precipitation. This is because [3-APTMS] was decreased by a factor of 100.
The data collected thus far shows strong evidence of amino group incorporation into the inorganic surface matrix, but further experiments, such as zeta potential, will be a stronger source of evidence of full amino surface saturation. NP11 shows a great starting point for future experiments varying concentrations of reactants. LbL deposition of this particle with negatively charged DNA might be a possible method of endocytic trafficking of genetic material. Other possibilities may include LbL of the negatively charged nanoparticle with rapid injection into a positively charged polymer solution. This positively charged nanoparticle surface could then be injected into DNA providing a stronger ionic interaction than incorporating 3-APTMS into the matrix crosslinks.
As show from the data precipitation and gelation are major problems encountered during CPNPs formation. Based on these results, decreasing the concentration TMOS to a calculated number, and doing an assay decreasing the concentration of TMAOH used could help increase 3-APTMS concentrations incorporated without an increase in particle radius. Consequently a decrease in precipitation and gelation will be expected.
Consistency in the preparation of CPNPs formed may have been one of the biggest challenges affecting this experiment. Towards the final trials many variables were identified and kept constant, such as the same stir bar used with the same RPM and the exactly same vials used. All vials were covered with aluminum to prevent degradation by light and purification by dialysis was completed within a time range of 24 to 48 hours. Rapid injection was controlled by always injecting the polymer solution with the micropipette tip about a mm into the solution from the surface. Variables that could not be controlled include the degradation of the solid polymer in storage, the degradation of CPNPs once formed, humidity and room temperature.
The author acknowledges Jonathan M. Behrendt from University of Manchester for providing the polymer P5, the paper titled Hybrid inorganic–organic composite nanoparticles from crosslinkable polyfluorenes, which provided the background information and procedures needed for this research, and special help regarding P5 solubility, degradation, and stability.
Table 1: Main Results
Conditions that Differ from Original Procedures
UV Abs = 0.030
PLQY =37.83734445 %
24 μM TMAOH
Abs = 0.022
PLQY = 29.8 %
3.75 μL 5.2 M 3-APTMS
Vigorous precipitation and gelation
3.75 μL 5.2M 3-APTMS and 24 μM TMAOH
Some precipitation and gelation
3.75 μL 0.52M 3-APTMS
3.75 μL 0.052M 3-APTMS
UV Abs = 0.017
PLQY = 17.3%
3.75 μL 0.0052M 3-APTMS
UV Abs = 0.0034
PLQY = 3.33%
3.75 μL 0.052M 3-APTMS
7.5 μL pure TMOS
3.75 μL 0.052M 3-APTMS
750 μL pure TMOS
75 μL of 0.0672 M TMOS and 3.75 μL of 5.2 M 3-APTMS
75 μL of 0.00672 M TMOS and 3.75 μL of 5.2 M 3-APTMS
UV Abs = 0.020
PLQY = 6.86 %
75 μL of 0.000672 M TMOS and 3.75 μL of 5.2 M 3-APTMS
UV Abs = 0.0072
PLQY = 2.49 %
Surface area of CPNP no TMOS: A == 58,106.89772 nm2= 58,000 nm2
Volume of TMOS per molecule =
TMOS radius = =
Area of TMOS per molecule =
Ratio of TMOS molecule per P5 NP = = 61,030.24653
Figure 2 6:
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