Tuesday, June 6, 2017

PolySciTech PLGA-NH2 used in study on nanoparticle biotransport: Nanoparticle transport explained using a kitten and a football game.


Nanoparticles are small… really small. To put it in perspective, a typical human cell ranges in size from 30-100 um in diameter. Nanoparticles range in size from 1 um down to 0.001 um (0.1 um being common) which means that a typical cell is between 300-1000 times the size of a nanoparticle. To put that in perspective, if a nanoparticle was the size of a kitten (~30 cm) then a human cell would close to the size of a football field (~90-100 M). One important question in science is how to get the kitten onto the football field… er… I mean… how to get the nanoparticle into the cell. This is important because nanoparticles can be loaded with medicines that have a variety of therapeutic effects which can be leveraged only if the nano-kitten can make it onto the cellular football field to make the game-winning kick. There’s two basic ways for either to happen. 1. Active targeting: For our metaphor we’ll assume nano-kitty has a game-day ticket which he politely presents at the front gate for entrance. Similarly, nanoparticles can be specifically conjugated to a specific signal molecule that has the right configuration to allow the nanoparticle access through the cellular membrane by accepted channels. (or) 2. Passive-targeting: This simply relies on the really small nano-kitten simply squeezing through a fence and slipping onto the football field ‘unseen’ due to its small size. Similarly, nanoparticles can sometimes enter cells simply because of their small size. A recent study using PST polymers focused on examining the processes at play in passive-targeting. In addition to final-products used directly for research, PolySciTech (www.polyscitech.com) provides a wide array of intermediates which can be used as precursors for making the final materials. For example, amine-endcap activated PLGA-NH2 has the capability to be chemically conjugated to a wide variety of molecules using common laboratory techniques such as carbodiimide-type conjugation between the PLGA-amine and a NHS-activated carboxylic acid on the other molecule. Recently, researchers at Johns Hopkins University utilized PLGA-NH2 (PolyVivo Cat# AI051) as part of an investigation into nanoparticle transport into living cells. They took the PLGA-NH2 and conjugated on a ‘caged’ rhodamine dye that did not fluoresce until it was prepared to do so by exposure to UV-light. This clever technique allowed the researchers to encapsulate the dye completely within the nanoparticles and precisely track and characterize the nanoparticles during cellular uptake studies. This research holds promise to improve nanotherapeutic formulations for treating a wide-variety of diseases. Read more: Schuster, Benjamin S., Daniel B. Allan, Joshua C. Kays, Justin Hanes, and Robert L. Leheny. "Photoactivatable fluorescent probes reveal heterogeneous nanoparticle permeation through biological gels at multiple scales." Journal of Controlled Release (2017). http://www.sciencedirect.com/science/article/pii/S0168365917306302

“Abstract: Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales – from tens of nanometers to tens of micrometers – using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample using brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. These findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.Keywords: Photoactivation; Fibrin; Cystic fibrosis; Nanoparticle; Drug delivery; Particle tracking; FRAP; Fluorescence microscopy; Diffusion. (Dye Conjugation protocol): Caged rhodamine-NHS ester and PLGA-NH2 were conjugated through formation of an amide bond. (Conjugating the dye to polymer in this way, rather than encapsulating the dye in the particles, reduces the likelihood of free dye being released.) Briefly, 90 mg of PLGA-NH2 was added to 5 mg of caged rhodamine-NHS ester, leading to a slight molar excess of dye compared to PLGA, 1.23:1, and put under vacuum for 1 h. The mixture was then flushed with nitrogen gas, dissolved in 500 μl of anhydrous dichloromethane (DCM), and reacted for 12 h at room temperature under nitrogen gas. Additional DCM was added as needed to facilitate transfer into 10 ml of − 20 °C diethyl ether to precipitate the product. The PLGA, now conjugated with the caged rhodamine, was washed twice in cold ether by centrifugation. Excess ether was decanted off and the final product, the purified PLGA-caged rhodamine, was placed in a lyophilizer (FreeZone 4.5 Plus; Labconco) for 12 h. The dried product was stored at − 20 °C in a shielded container to prevent exposure to incident UV light. It is important to note that the amide bond is formed between the amine end-cap of the PLGA and the succinimidyl (NHS) ester on the rhodamine; that is, the dye itself and not the ortho-nitroveratryloxycarbonyl (NVOC) cage is directly conjugated to PLGA. Thus, when the photolytic reaction occurs upon exposure to UV light, only the caging group is cleaved, and free dye is not released”
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