Langmuir. 2016 Aug 9;32(31):7929-42.

Mechanistic Studies on the Self-Assembly of PLGA Patchy Particles and Their Potential Applications in Biomedical Imaging.

Salvador-Morales C1,2, Brahmbhatt B1,2, Márquez-Miranda V3, Araya-Duran I3, Canan J4, Gonzalez-Nilo F3, Vilos C3,5, Cebral J1,2, Mut F1,2, Lohner R6, Leong B7, Sundaresan G7, Zweit J7.
  • 1Bioengineering Department, George Mason University , 4400 University Drive, MS 1G5, Fairfax, Virginia 22030, United States.
  • 2Krasnow Institute for Advanced Study, George Mason University , 4400 University Drive, MS 2A1, Fairfax, Virginia 22030, United States.
  • 3Center for Bioinformatics and Integrative Biology, Facultad de Ciencias Biologicas, Universidad Andres Bello , Santiago, Chile 8370146.
  • 4Fundación Fraunhofer Chile Research , M. Sanchez Fontecilla 310, Las Condes, Chile 7550296.
  • 5Center for Integrative Medicine and Innovative Science, Faculty of Medicine, Universidad Andres Bello , Santiago, Chile 8370146.
  • 6Center for Computational Fluid Dynamics, College of Sciences, George Mason University , Fairfax, Virginia 22030, United States.
  • 7Center for Molecular Imaging, Department of Radiology, Virginia Commonwealth University , Richmond, Virginia 23298, United States.

Abstract

Currently, several challenges prevent poly(lactic-co-glycolic acid) (PLGA) particles from reaching clinical settings. Among these is a lack of understanding of the molecular mechanisms involved in the formation of these particles. We have been studying in depth the formation of patchy polymeric particles. These particles are made of PLGA and lipid-polymer functional groups. They have unique patch-core-shell structural features: hollow or solid hydrophobic cores and a patchy surface. Previously, we identified the shear stress as the most important parameter in a patchy particle’s formation. Here, we investigated in detail the role of shear stress in the patchy particle’s internal and external structure using an integrative experimental and computational approach. By cross-sectioning the multipatch particles, we found lipid-based structures embedded in the entire PLGA matrix, which represents a unique finding in the PLGA field. By developing novel computational fluid dynamics and molecular dynamics simulations, we found that the shear stress determines the internal structure of the patchy particles. Equally important, we discovered that these particles emit a photoacoustic (PA) signal in the optical clinical imaging window. Our results show that particles with multiple patches emit a higher PA signal than single-patch particles. This phenomenon most likely is due to the fact that multipatchy particles absorb more heat than single-patchy particles as shown by differential scanning calorimetry analysis. Furthermore, we demonstrated the use of patchy polymeric particles as photoacoustic molecular probes both in vitro and in vivo studies. The fundamental studies described here will help us to design more effective PLGA carriers for a number of medical applications as well as to accelerate their medical translation.

PMID: 27468612; DOI:10.1021/acs.langmuir.6b02177

 

Supplement:

Summary: Targeted drug delivery based on the use of polymeric nanoparticles is one of the most promising examples of personalized medicine. However, several technical challenges have prevented us from reaching clinical settings with this engineering approach. At the fundamental level, a major limitation has been to understand in depth the self-assembly process that drives the formation of nano-size and micron-size carriers, as well as to design their surface chemistry adequately in order to achieve an appropriate immune response. Moreover, the self-assembly process that occurs during the nanoprecipitation, single or double emulsion methods, the most commonly used methods to synthesize these particles, is different at the nano-size and micron-size scales. Furthermore, the self-assembly process depends on several factors, including mechanical and chemical parameters such as shear stress and solvent composition, among others. Often, all of these fundamental aspects are not fully studied in detail during the particle synthesis. Thus, thoroughly addressing these fundamental issues is essential to reach clinical settings with these novel nanomaterials. Among different types of polymeric nanoparticles, PLGA nanoparticles have previously been tested in Phase I clinical trials, and have shown high efficacy and low toxicity [1]. Therefore, a significant contribution of Nanomedicine to society will be achieved if this type of nanoparticle is approved by the FDA in the upcoming years, as it will revolutionize the way medical oncologists proceed in treating cancer patients.

 

In the last 15 years, a large number of particle designs has been reported in literature. Our research group reported in 2012 a new type of anisotropic particles that we named as patchy polymeric particles (PPP) as shown in below figure [2-3]. Since then, we have been studying the formation of these particles and exploring their applications in several sub-fields in medicine.  Patchy polymeric particles (PPP) have anisotropic surface domains that can be remarkably useful in diverse medical and industrial fields because of their ability to simultaneously present two or more different surface chemistries on the same construct. These new patchy particles are able to execute two or more functions simultaneously through the patch or patches and through the particle’s core. The fact that patches with different surface chemistries can be formed on the particle’s surface allows us to incorporate different organic or inorganic molecules according to the medical need. For example, the patches can be further functionalized with organic or inorganic molecules to target or image different cells and tissues. The functionalization of particles using a patch is greatly advantageous in several ways. For instance, the patch allows us to cluster a high number of ligands, which can then be taken up more easily by cells, organs, or tissues. Next, the clustering of exogenous photoacoustic contrast agents can greatly enhance an imaging signal.

 

In this paper, we summarize the most recent developments on the mechanisms involved in the formation of PPP. Also, this paper reports the exciting imaging application of PPP both in in vitro and in vivo studies. In this study, we used an integrated computational and experimental approach to fully elucidate the unique external and internal structure of PPPs. The computational approach included the development of seminal computational fluid and molecular dynamics simulations to understand the external and internal properties of PPPs. Both Computational Fluid Dynamics (CFD) simulations and experiments concur that shear stress is the most important parameter for the formation of the external and internal structure of PPPs. Single-patch particles have a hollow core whereas multi-patch particles have a solid core. Furthermore, CFD simulations help us to tune the thickness of the particle’s shell. Molecular Dynamics (MD) simulations revealed the formation of a lipid bilayer on the particle’s surface and therefore gave insights about the arrangement of Lipid-PEGylated functional group on the particles’ surface. This is the second most important polymer used in the PPP’s synthesis. This information is very critical as the carrier’s surface chemistry determines its fate in animals and humans. Additionally, MD demonstrated the hollow nature of single-patch particles, which matched with our experimental findings.

 

In this paper, we also reported the intrinsic photoacoustic (PA) properties of SPPP [4].  We described a method to enhance the intrinsic PA of PPP by incorporating a semiconducting polymer in their core. We functionalized the particle’s surface with folic acid and assessed its targeting efficacy in KB3-1 cervix carcinoma cells. We observed that targeted SPPP are avidly taken up by cancer cells. These particles were localized in the nucleus and cytoplasm of the cell. Moreover, we tested the PA performance of SPPP and compared it with gold nanorods using standard PA contrast agents. We observed that SPPP emit a PA signal comparable to that of gold nanorods. Furthermore, we conducted animal experiments in which we implanted cancer tumors in female mice and assessed the targeting and PA performance of the engineered SPPP. The results from animal studies show a significant presence of SPPP in the tumor site, which allowed us to detecting them with the photoacoustic imaging technique. Further experiments are underway to investigate the therapeutic effect of SPPP by encapsulating a chemotherapeutic drug in the particle core and releasing it at the tumor site. It is worth notice that PPP of a couple of microns in size can be taken by breast cancer cells as shown in below figure.

 

 

 

Importance of the Study: This study is novel in the field of cancer nanotechnology for several reasons. First, we conducted in depth mechanistic studies on the self-assembly process between PLGA and Lipid-PEGylated functional groups. These studies are essential to highly control the physicochemical properties of these particles. Our molecular dynamics simulations show that van der Waals interaction are the most prominent chemical interaction that exists at the molecular level between these two polymers. Second, this study expands upon the knowledge that the shear stress determines both the internal and external morphology of patchy polymeric particles. We conducted a highly detailed characterization of these particles, which allows us to optimize the key parameters that provide the best yield of patches. Thus, this study will help us to fine-tune several important external and internal features of the polymeric particles including number of patches, nature of the core: solid or hollow, thickness of the particle’s shell, and type of surface functionalization. Furthermore, the in in vitro and in vivo experiments conducted in this study also showcased the feasibility of these particles as promising photoacoustic molecular probes. The ability of these particles to provide an imaging outcome at different stage of cancer treatment will help medical oncologists to decide the best course of action to maximize efficacy and minimize toxicity. Finally, the development of seminal molecular and computational fluid dynamic simulations will help us to achieve greater batch control and scalability. These are factors that will ensure the successful medical translation of patchy polymeric particles in the long-term.

 

REFERENCES

  1. J. Hrkach, D.V.Hoff, M. M. Ali, E. Andrianova, J. Auer, T. Campbell, D.D.Witt, M. Figa,  M. Figueiredo, A. Horhota, S. Low, K. McDonnell,  E. Peeke, B. Retnarajan et al. Preclinical Development and Clinical Translation of a PSMA-Targeted Docetaxel Nanoparticle with a Differentiated Pharmacological Profile. Science Translational Medicine, 4, 128ra39, 2012.
  2. C. Salvador-Morales, P.M.Valencia, W. Gao, R. Karnik, P. Farokhzad. Spontaneous Formation of Heterogeneous Patches on Polymer-Lipid Core-shell Particle Surfaces during Self-Assembly, Small, 1-7, 2012.
  3. N. Rasheed, A.A. Khorasani, J. Cebral, F. Mut, R. Löhner, C. Salvador-Morales. Mechanisms involved in the formation of biocompatible lipid polymeric hollow patchy particles. Langmuir, 3, 6639-6648, 2015.
  4. C. Salvador-Morales, B. Brahmbhatt, V. Márquez-Miranda, I. Araya-Duran, J. Canan, F. Gonzalez-Nilo, C. Vilos, J. Cebral, F. Mut, R. Lohner, B. Leong, G. Sundaresan, J. Zweit. Mechanistic Studies on the Self-assembly of PLGA Patchy Particles and their Potential Applications in Biomedical Imaging. Langmuir, 32, 7929-7942, 2016.

 

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann