Layer-by-Layer Deposition of Highly Fluorescent Inorganic Conjugated Polymer Nanoparticles for Encapsulation of Hydrophobic Drugs, Cellular targeting, Bio-Stability and Increased Drug Efficacy

Author: Camilo M Hernandez

Introduction:

In the past decade, Layer-by-Layer (LbL) deposition, a nanoscale film fabrication technique[1], has been the focus of interest for nanoparticle (NP) drug[2] [3] and gene[4] [5] delivery by researchers. Post formation surface modifications of NPs can include side chain covalent modifications due to reactions with functional groups[6], or LbL deposition of electrostatic interactions between alternatingly charged polyelectrolyte layers[7]. There is evidence that incorporating targeting moieties improves NP uptake[8] due to cell ligand binding and improves residence time in tumors[9], so it is of great importance for researchers[10] to find novel post formation surface modifications that are easy to mass-produce and easily exchangeable to access specific cells.

LbL deposition of polymers can help control certain aspects of the nanoparticles dimension, such as decrease nanoparticle radius and surface charge, as well as increase solubility, half-life in circulation and cellular uptake[11]. By using LbL techniques an experimenter can also control undesirable burst drug release associated with polymeric nanoparticles due to reducing the permeability of the nanoparticle with increasing layers and diffusion length which the drug had to cross before being released. LbL nanoparticles have the ability to sheath layers according to pH of the environments, making them capable of adapting to different environments[12]. A shielding de-shielding mechanism triggered by the low pH of hypoxic environment would be key for cancer therapies. Also, a final antifouling layer on the LbL coated NPs can provide a stealth coating that will avoid rapid reticuloendothelial system (RES) clearance[13] This will allow NP accumulation in tumor interstitials due to the enhanced permeation and retention effects[14] (EPR).

The nanoparticle core may be composed of a drug nanoparticle (such as an anticancer drug) [15] solid lipid polymer nanoparticle (SLNP)[16], conjugated polymer nanoparticle (CPNP)[17], inorganic nanoparticles (INPs)[18] or a metal core nanoparticle such as gold (AuNPs). Highly fluorescent polymers, in the recent years, have been used as a tool for biomedical applications, such as bioimaging[19], biosensing[20], precise timed release of biological molecules[21], and in tissue engineering[22] Conjugated polymers have shown great promise as drug delivery methods because of the advantage of having higher backbone hydrophobicity, low cytotoxicity, increased photoluminescence quantum yields (PLQYs)[23], increase in bio stability and the ability to undergo a more versatile range of surface modifications[24] as opposed solid lipid nanoparticles which have also been used for the delivery and transport of hydrophobic drugs to targeted cells, but are limited due to processes such as endocytic recycling and high excretion rates from the circulatory system by macrophages[25].  Inorganic nanoparticles have shown to great promise as nanoparticle cores due to higher thermal stability and a multitude of tunable optical properties11. Additionally, in the preparation of polyelectrolyte capsules, core destruction is generally more problematic in case of polymeric colloids when compared to inorganic polymers because of adsorption of core polymer onto the shell layers during core dissolution leading to modification of the layer properties[26].  

In this experiment, hybrid inorganic conjugated polymer nanoparticles (iCPNPs) will be considered as the nanoparticle cores of study. The merging of these two kinds of polymers may hold great promise in finding a novel and widely tunable drug carrier. iCPNPs are made from conjugated polymers containing an inorganic pedant charged group such as silica[27]. These pedant polymerizable charged groups can provide a covalent surface modification between the CPNP and the inorganic charged host matrix, giving the nanoparticle core a surface charge[28]. NPs must have a core host surface charge before LbL deposition can be done for electrostatic interaction to be sufficiently strong to prevent dissolution of layers. iCPNPs covalently bonded to a charged silica matrix will contain a negative surface charge where electrostatic interaction for layering may begin.

For over a decade researchers have reported that an initial layer of poly-L-Lysine (PLL) improves cellular uptake of molecules[29] [30].  Researchers part of P. T. Hammond’s research group have found that by using modified PLL with iminobiotin in the first layer, a second layer composed of neutravidin (nav) can bridge the PLL and biotin end-functionalized poly(ethylene glycol) (PEG) third layer via neutravidin-iminobiotin bonds12.  Iminobiotin and neutravidin are modified versions of biotin and avidin respectively.  PEG has been shown to be a powerful antifouling polymer that would enable the layered nanoparticles to avoid rapid RES clearance and a greater EPR13-14.The iminobiotin-neutravidin interaction is a pH dependent non-chemical bond allowing the removal of the third PEG “shielding” layer upon contact with the low pH of the hypoxic tumor environment[31], allowing for the PEG layer to become exposed and allow cellular uptake. The iminobiotin-neutravidin bond is most stable at a pH above 8.0 but still shows a slow degradation at pH 7.4[32].  Approaches sensitive to pH have already been shown to be therapeutically more advantageous than conventional pH-insensitive methods[33]. Hypoxia and acidosis are reasons that tumor cells gain resistance to chemotherapy and radiation34-[34], which gives an advantage to pH sensitive therapies that sensitize hypoxic tumor cells to treatment[35]-[36].

To date, conjugated polymers fall into four notable categories[37]: poly(fluorene) (PF), poly(p-phenylenevinylene) (PPV), poly(p-phenyleneethynylene) (PPE), and poly(thiophene) (PT). In this experiment, PF and PPE conjugated polymers will be tested for their ability to form LbL nanoparticles and traffic drugs, siRNA, and their ability to undergo core destruction.  Cationic Conjugated polymers have also been tested as potential delivery methods. Moon et. Al. have shown the possibility of siRNAs delivery and targeted gene knockdown in plant protoplasts, using amine containing PPE nanoparticles[38]. By incorporating cationic conjugated polymers (cCPs) into the first layer of the iCPNP, siRNA can become trafficked into the cell, and monitored in-vivo.

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[39] [40]. In this experiment the second method will be explored, because of its ability to make smaller (10-200 nm), more monodispersed CPNPs[41].

Research Objectives, Design, and Methods:

The goal of the proposed research is to develop inorganic conjugated polymer nanoparticles using LbL techniques for the detection and treatment of cancer.  To complete this research goal, the following tasks have been proposed for this goal.

  1. Preparation of PF iCPNPs.

The first objective is synthesis synthesis of PF iCPNPs, and will be followed using procedures from Behrendt, Jonathan M., et al27 shown in scheme 1. The iCP used is refered to as polymer 5 in the paper. A 2000-ppm polymer solution consisting of 20 mg of P5 in 10 g of THF will then be made. After dissolving the polymer by stirring for 48 hours in the dark, excess insoluble matter will be filtered out using a 0.45 nm PTFV filter resulting in a solution of unknown decreased concentration. The actual concentration will be determined by lyophilization of the solution. The nanoparticles inorganic matrix will be formed under tetramethyl orthosilicate (TMOS) to polymer weight ratios of 0:1, 5:1, and 10:1. To 750 μL of this polymer solution, 75 μL of pure THF, 0.68 M TMOS in THF, or 1.4 M TMOS in THF will be added with stirring for 5 minutes respectively. Then, the polymer solution will be rapidly injected into 3 mL of 24 mM TMAOH in 18 Ω DI water with stirring for 5 minutes. The resulting nanoparticle solution will be transferred to dialysis chamber (2 kDa MWCO) and flushed with 1 L of water. Resulting purified nanoparticle solutions will be filtered using P8 filter paper and stored in lightproof vials. TMOS will be diluted in THF prior to aggregation. Tetramethylammonium hydroxide (TMAOH) in water will be used as a surfactant upon nanoparticle aggregation incorporating the TMOS matrix. CPNPs will then be characterized by absorption and emission spectrophotometry, zeta potential and nanoparticle radios.

Scheme 1:

 

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  1. Preparation of PPE iCPNPs:

PPE iCPNPs will be formed by following scheme 2. 2,5-diiodobenzene-1,4-diol will be reacted 1-bromoheptane with K2CO3 in methanol for one hour at room temperature to form 1,4-diiodo-2,5-bis(octyloxy)benzene (1).

1 will then be reacted with TMS-acetylene to form 1,4-diethynyl-2,5-bis(octyloxy)benzene (2). 2 will then be reacted with potassium carbonate in methanol to from triethoxy(3-isocyanatopropyl)silane. Then, 2,5-diiodobenzene-1,4-diol will also be reacted with 6-bromohexanol and potassium carbonate in methanol to form 6,6'-((2,5-diiodo-1,4-phenylene)bis(oxy))bis(hexan-1-ol) (4). 4 will then be reacted with triethoxy(3-isocyanatopropyl)silane at room temperature for sixteen hours under triethyl amine dissolved in DCM to form 5. Sonogashira coupling[42] with PdCl2(PPh3)2 , CuI, in DMF and triethyl amine will then be employed to produce the PPE iCP (6) from 3 and 5. After purification of the polymer solution, the nanoparticles inorganic matrix will be formed under tetramethyl orthosilicate (TMOS) to polymer weight ratios of 0:1, 5:1, and 10:1. To 750 μL of this polymer solution, 75 μL of pure THF, 0.68 M TMOS in THF, or 1.4 M TMOS in THF will be added with stirring for 5 minutes respectively. Then, the polymer solution will be rapidly injected into 3 mL of 24 mM TMAOH in 18 Ω DI water with stirring for 5 minutes. The resulting nanoparticle solution will be transferred to dialysis chamber (2 kDa MWCO) and flushed with 1 L of water. Resulting purified nanoparticle solutions will be filtered using P8 filter paper and stored in lightproof vials. TMOS will be diluted in THF prior to aggregation. TMAOH in water will be used as a surfactant upon nanoparticle aggregation incorporating the TMOS matrix. CPNPs will then be characterized by absorption and emission spectrophotometry, zeta potential and nanoparticle radius.

Scheme 2:

A:

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B:

 Macintosh HD:Users:camilohernandez:Desktop:Screen Shot 2014-04-29 at 11.58.11 AM.png

  1. Preparation of Cationic CPs (cCPs):

Cationic CPNPs will be synthesized following procedures from Ko Yoon-Jo et. Al.[43] following procedures from the paper shown here in scheme 3.

Scheme 3:

Macintosh HD:Users:camilohernandez:Desktop:Screen Shot 2014-04-29 at 11.12.16 AM.png

Macintosh HD:Users:camilohernandez:Desktop:Screen Shot 2014-04-29 at 11.12.24 AM.png

  1. Preparation of Iminobiotin Functionalized PLL:

Pol-L-lysine (PLL) (15 kDa) will be functionalized to NHS-activated iminobiotin or NHS-activated biotin will be reacted in aqueous conditions at a pH of 8.0 for 2 hours and then dialyzed using a 5 kDa dialysis bag. The reaction feed ratio will be calculated on about 20% of the primary amine side groups.

  1. LbL formation on nanoparticles.

Materials that will be used are as followed: The PF iCPNP from objective 1, PPE iCPNP from objective 2, the cCPs from objective 3, siRNA coding for a green fluorescent protein (GFP), Imminobiotin functiolized PLL (IB-PLL) from objective 4, dextran sulfate (15 kDa), mPEG-biotin (20 kDa), and neutravidin. LbL nanoparticles will be prepared using a commonly used technique used by Gittins DI and Caruso F.[44] Table 1 shows the possible combinations of Lbl possible. For each layer added, nanoparticles will be mixed with a saturating amount of layering material with continuous agitation for 2 h, followed by particle purification with three centrifugation and re-suspension cycles. The centrifugation periods will be performed at 30000 rcf for 2 hours. The re-suspension cycles will be performed in Millipore water at a pH of 7.4 except for situations involving iminobiotin, where a pH of 8.0 will be used instead. After LbL formation has been performed, core dissolution will be perforfed following procedures from Gittins DI and Caruso F44.

 

Core

1st Layer (L1)

2nd Layer

(L2)

3rd Layer

(L3)

4th Layer

(L4)

5thLayer

(L5)

NP1

iPF

IB-PLL

neutravidin

mPEG-biotin

NP2

iPPE

IB-PLL

neutravidin

mPEG-biotin

NP3

iPF

cCP

siRNA

IB-PLL

neutravidin

mPEG-biotin

NP4

iPPE

cCP

siRNA

IB-PLL

neutravidin

mPEG-biotin

  1. Characterization of iCPNPs:

Resulting LbL iCPNPs will be characterized using absorption and emission spectrophotometry, surface zeta potential, and dynamic light scattering. UV-visible absorption spectra will be recorded using a Varian Carry 50 Bio UV-visible spectrophotometer, in water at room temperature (25 oC). Fluorescence spectra and PLQY will recorded and calculated using a Horiba Jobin Yvon Fluorolog Model FL-1039/40, in water at room temperature (25 oC). The size and Zeta potentials of the nanoparticles will be measured using a Malvern Zetasizer Nano S, at room temperature, in 18  deionized water. For the zeta measurements 700 of the sample will be used per measurement for a total of 6 measurements, and for size measurements 3 measurements of 100 mL will be used.  

  1. Cellular Studies and Transfection Efficiency:

Cellular studies on cultured cancer cells, will be performed and a toxicity assay and colocalization will be performed. Exact cells worked on will be hot cell cultures. LbL iCPNPs transfection efficiency will be calculated by using counting the number of fluorescent cells in a given area through microscopic observations. siRNA delivery efficiency will be calculated by calculating the amount of cultured cells displaying GFP.

Research Implications:

Finding a versatile drug delivery system capable of loading a versatile range of drugs and capable of adapting to different environments would be one of the greatest achievements for pharmaceutical engineering and biomedicine. This will bring about a revolution in medicine due to the ability to deliver gene therapies to a range of mutated cells. Tumors inaccessible by conventional medicine, or invasive surgery, will be treatable, giving terminal ill patients a new chance at life. These methods may be incorporated into all type of gene delivery techniques, curing hereditary diseases and disadvantageous random mutations.

Research Timeline:

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The time scale is highlighted for the 12 week summer term. Black slots are on time, while green are early and red are late to meet deadlines.

Sources:


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