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Manufacture Technology and Equipment, Small Molecules

Layer by Layer

The fabrication of nanoparticles sounds complex enough by itself, so what about when we try to be even more adventurous, such as fabricating hollow nanocapsules? How would you feel about fabricating nanoreactors or designing small nanoparticles with two distinct chambers? Innovative ideas are sometimes very complicated, but luckily handy tools are out there to help. In particular, we strongly believe that the sequential deposition of molecular layers, widely known as Layer-by-Layer (LbL) technology holds great potential. Since its introduction by Decher and colleages in 1992 (1), coating flat surfaces, as well as micro- and nanoparticles, using LbL technology has become an active area of research that has many potential applications.

In its simplest form, LbL is based on alternating the deposition of oppositely charged polyelectrolytes on a charged surface. For example, a negatively charged surface is coated with a positively charged polyelectrolyte. Polyelectrolytes are polymers with charged repeating units; when they are adsorbed by electrostatic attraction, some charges will be neutralized and some will remain free, to which a second layer of negatively charged polyelectrolyte is adsorbed. This process can be repeated as much as needed to build up a layered system of tunable characteristics. Interestingly, LbL assembly can be done using other types of interaction as well, such as hydrogen bonding, coordination bonding, and hydrophobic interactions. As a result, a diverse range of components is at hand to build LbL structures, including – but not limited to – synthetic and natural polymers, proteins, nucleic acids, dyes and dendrimers (2).

Perhaps we have made LbL deposition sound very simple (and, in some ways, it is), but when it comes to creating very complicated, multifunctional structures the magic only happens when the right components are added in the right sequence. Initially, LbL structures were typically flat films coating charged surfaces; for example, surgical stents coated with films that release anti-bacterials to reduce infection-related complications or the immobilization of antibodies to create immunosensors (3, 4). A turning point was the introduction of the “sacrificial core” technique. Let’s consider a suspension of calcium carbonate nanoparticles carrying a negative charge. After coating with several polyelectrolyte layers, cores are destroyed using an EDTA solution, leaving a suspension of hollow nanocapsules. If these layers were assembled on enzyme crystals followed by dissolution of the enzyme crystals, we can obtain high enzyme loading in nano-sized capsules. As small molecules can diffuse through the pores of the LbL shell, we obtain nanoreactors – where substrates can diffuse in, get processed by the enzyme and products diffuse out (5).

Enzymes were also used to design multilayer films that are glucose sensitive, releasing insulin in response to the presence of glucose molecules in the surrounding medium. The design depends on an assembly of alternating layers of glucose oxidase (GOD) and catalase (CAT) on positively charged insulin crystals salted out in an excess of sodium chloride. GOD converts glucose into gluconic acid, releasing molecular oxygen and producing H2O2. The production of gluconic acid lowers the pH value at the surface of the shells and enhances its permeability to release insulin. In addition, the decrease in pH favors higher insulin solubility in water, thus facilitating the release from the system. Nevertheless, GOD activity may suffer decay with time due to peroxide-introduced degradation, leading to low sensitivity to glucose. However, the role of CAT is to convert the aggressive H2O2 into H2O and O2 – most of the oxygen produced is consumed by GOD (6).

Another exciting (but complex) application of LbL technology is the co-delivery of protein and small-molecule drugs via double chambered nanocarriers, with the aim of overcoming the non-uniform distribution of different, simultaneously administered drugs. In an interesting example, liposomes loaded with chemotherapeutic agents were coated with LbL films containing siRNA molecules to attack an aggressive form of breast cancer (7).

LbL is a technology with high potential, and with many research groups in this field, we expect to see a plethora of applications. But we should not forget that technical challenges exist in terms of upscaling the process and the handling of excess volumes of liquids. However, these are challenges that we can overcome together with the pharma industry – with the ultimate goal being to introduce new, innovative and more efficient products.

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  1. G. Decher, J.D. Hong and J. Schmitt, “Build-up of ultrathin multilayer films by a self-assembly process. III. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces,” Thin Solid Films 210-211, 831–835 (1992).
  2. K. Ariga, J.P. Hill and Q. Ji, “Layer-by-layer assembly as a versatile bottom-up nanofabrication technique for exploratory research and realistic application,” Phys. Chem. Chem. Phys., 9(19), 2319-40 (2007).
  3. A. Shukla et al., “Controlling the release of peptide antimicrobial agents from surfaces,” Biomaterials 31(8), 2348-57 (2010).
  4. O.S. Sakr and G. Borchard G. 2013. “Encapsulation of Enzymes in Layer-by-Layer (LbL) Structures: Latest Advances and Applications,” Biomacromolecules 14, 2117−2135 (2013).
  5. X. Cui X et al., “Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing,” Biosens. Bioelectron., 18(1), 59-67 (2003).
  6. W. Qi et al., “Triggered release of insulin from glucose-sensitive enzyme multilayer shells,” Biomaterials, 30(14), 2799-806 (2009).
  7. Z.J. Deng et al., “Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for potential Triple-Negative Breast Cancer Treatment,” ACS nano., 7(11), 9571–9584 (2013).
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