How to Characterize and Purify Your Polymer Nanoparticles

1. How to Characterize and Purify Your Polymer Nanoparticles CD The use of polymers are increasing because of their diverse physical and chemical properties and extensive functions (Table 1). For example, polymer nanoparticles can be used to deliver cancer drugs to specific sites. The biomedical field has experienced tremendous growth over the past few decades. Clinical studies of polymer-protein conjugates and polymer-drug conjugates have been conducted successfully and regulatory approvals have been obtained (Figure 1). Table 1. Classification, application areas, advantages, and disadvantages of polymer-based nanomaterials Classification Materials Application Areas Advantages Disadvantages Hemostasis material, medical dressing, hydrogel, drug delivery carrier, gene transfer Biocompatibility, antimicrobial, innocuous, easily degradable, adsorbability, film formation Poor spinnability, poor strength, lowwater-solubility Chitosan Poor mechanical properties, resistance to water, poor blocking performance Hemostasis material, tissue- engineered scaffold, drug delivery carrier, bone repair material Extensive sources, low price, degradation products safe and non-toxic, non-antigenic Natural polymeric material Starch Hypotoxicity, biocompatibility, suppresses tumor growth, enhances immunity Pharmaceutical excipient, pepcid complete, medical dressing Bad biodegradability, cell attachment poor Alginate Cellulose Pharmaceutical adjuvant Extensive sources, low price Rare adverse reactions (continued) Email: info@cd-bioparticles.com Tel: 1-631-633-6938

2. Table 1. (continued) Poly β- hydroxybutyrate (PHB) Biodegradable, safe, non-toxic, good physical and chemical properties Biosynthesis material Drug-delivery carrier, tissue engineering material High crystallinity, bad thermal stability Anti-adhesion materials, patch, drug-delivery carrier, bone-fixing device, suture, tissue-engineered scaffold Poor toughness, degradation speed slow, hydrophobicity, lack of reactive side chain groups Biocompatibility, good mechanical properties, safe, non-toxic Polylactic (PLA) Chemosynthes material (Copolymer) Low cost, rich resource, good mechanical properties Polyurethane Excipients, medical bandage Degradation speed slow Higher cost, drug-loading capacity and stability can be improved Poly(lactic-glycolic acid) (PLGA) Absorbable suture, drug delivery, bone screwfixation, tissue repair Controllable biodegradability, biocompatibility Polymethyl methacrylate resin (PMMA) Bone-fixation materials, dental materials, artificial crystal Easy operation, good biocompatibility Monomer has cytotoxicity, easy oxidation Figure 1. The applications of polymer-based nanomaterials in the production of vaccines and drugs. Regardless of the polymer composition, synthetic route, or nanoparticle type, reliable and relevant physicochemical characterization (PCC) is critical to the translation process of formulations and is an important requirement for regulatory approval. For example, particle size is known to affect biodistribution and clearance routes. Particle charge affects blood compatibility. Surface modification (or lack of surface modification) has been shown to affect biocompatibility and increase toxic side effects. A small change in any of these parameters may adversely affect the efficacy of the nano-drug; or worse, it will fatally increase toxicity. In addition, the stability of the formulation (i.e., storage stability, plasma stability, etc.) and batch-to-batch consistency must be thoroughly evaluated for a longer term. Because small changes in the properties of nanoformulations can have devastating effects, strict physical and chemical characterization will be necessary for successful clinical translation of nanomedicines. This is also a requirement in the regulatory process. Email: info@cd-bioparticles.com Tel: 1-631-633-6938

3. We will briefly introduce the most common analytical techniques used to evaluate these parameters, as well as the purification methods of polymer nanoparticles before their use in preclinical and clinical applications. Analytical Techniques GPC The gel permeation chromatography (GPC) is widely used to determine the molecular weight of materials dissolved in organic solvents as well as the physical stability of assembled nanomaterials. The nanomaterials are eluted as a function of their molecular weight: the larger the molecular weight, the faster the elution. The quantification of the eluted samples is performed by means of UV-Vis absorption or changes in the refractive index. • HPLC High-performance liquid chromatography (HPLC) is used to separate, identify, and quantify each component in a mixture. It relies on a pump to pass a pressurized liquid solvent containing a sample mixture through a column filled with a solid adsorbent material. The interaction of each component in the sample with the adsorbent material is slightly different, resulting in different flow rates for each component and separating the components as they flow out of the column. More interactions between the molecules and the column filling will delay the elution. In addition, the molecules elute in the characteristic pattern of each compound. It will produce a chromatogram with peaks for each compound. • SEC Size exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method that separates molecules in solution based on their size (sometimes on their molecular weight). It is usually applied to macromolecules or polymer complexes, such as proteins and industrial polymers. Generally, gel filtration chromatography uses an aqueous solution to transport a sample through a chromatographic column instead of using an organic solvent as the mobile phase, which is termed the gel permeation chromatography. The column is filled with tiny porous beads composed of dextran polymers, agarose, or polyacrylamide. The pore size of these beads is used to estimate the size of macromolecules. SEC is a widely used polymer characterization method because it provides good molar mass distribution (Mw) results for polymers. • FTIR Fourier transform infrared spectroscopy (FTIR) is an analytical technique used to identify organic (in some cases inorganic) materials. This technique measures the relationship between the absorption of infrared radiation and the wavelength of the sample material. The infrared absorption band recognizes the molecular composition and structure. When the material is exposed to infrared radiation, the absorbed infrared radiation usually excites the molecules into a higher vibration state. The wavelength of light absorbed by a particular molecule is a function of the energy difference between the at-rest and excited vibrational states. The wavelength absorbed by the sample is characteristic of its molecular structure. • Email: info@cd-bioparticles.com Tel: 1-631-633-6938

4. UV-Visible Spectroscopy Ultraviolet-visible light (UV-Vis) spectroscopy is one of the most popular analytical techniques because it is very versatile and can detect almost every molecule. Using UV-Vis spectroscopy, UV-Vis light passes through the sample, and the transmittance of the sample to light is measured. According to the transmittance (T), the absorbance can be calculated as A = -log (T). Thus, the absorbance spectra of a compound at different wavelengths is obtained. The absorbance at any wavelength is determined by the chemical structure of the molecule. UV-Vis can be used qualitatively to identify functional groups or confirm the identity of compounds by matching absorption spectra. It can also be used in a quantitative manner because the concentration of an analyte is related to absorbance according to Beer's law. • Fluorimetry Fluorimetry is a very sensitive spectroscopic technique. This technique involves a quantitative measurement of the fluorescent signal usually produced by aromatic molecules to detect and characterize organic and inorganic compounds by applying a fluorescent laser to the sample. This technology has been used in a variety of applications, all of which take advantage of the ability of the materials studied to be excited under a fluorescent laser and emit fluorescence at another wavelength. • DSC Differential scanning calorimeter (DSC) is a popular thermal analysis instrument used to measure the physical property and temperature changes of samples over time. In other words, the device is a thermal analysis instrument that determines the temperature and heat flow associated with the material transition as a function of time and temperature. During temperature changes, DSC measures the amount of heat that the sample over-radiates or absorbs based on the temperature difference between the sample and the reference substance. This is a useful technique to determine the structure and stability of nanomaterials and their conformation because the transitions of materials will change with the composition of nanomaterials. • TGA Thermogravimetric analysis (TGA) is the mass loss or gain in a sample as a function of temperature and time under controlled atmospheric conditions. More simply stated, TGA measures the mass lost or gained when heating a sample according to a predefined temperature and time program. Thermogravimetric analysis can be used to determine the properties and characteristics of the polymer, the decomposition temperature of the polymer, the moisture content or residual metal content of the sample. • Email: info@cd-bioparticles.com Tel: 1-631-633-6938

5. Technique Characteristics that analyses Advantages Disadvantages Rapid and simple High resolution High resolution Rapid and easy performance Low cost/sample Small sample volumes High resolution Rapid and simple Interaction sample with column flling GPC Molecular weight Quantification of actives Purification HPLC Interaction sample with column flling Interaction sample with column flling Need of a labelling tag Complicated sample preparation Interference of water Relatively low sensitivity Requires dried samples Destructive SEC Purification Fast and inexpensive Characteristic of each material Possible quantification FTIR Chemical composition Cost effective Simple and fast Useful for a variety of compounds High sensitivity Compound specificity Quantification of concentration Size and shape determination UV-Vis Interference between materials Quantitative determination fluorescence Limited to fluorescent compounds Limited fluorescence lifetime Fluorimetry Glass transition Melting temperature Low amounts of sample High precision and sensitivity Requires sample preparation Requires an appropriate reference DSC Low amounts of sample High precision and sensitivity Requires sample preparation Requires an appropriate reference TGA Weight loss Purification Techniques In order to ensure that the formulation is safe and free of contaminants, it is strongly recommended to carry out the purification step and then physical and chemical characterization before starting the preclinical and clinical analysis of the polymer nanoparticles. There are various methods for purification of colloidal nanomaterials, such as magnetic separation of magnetic nanoparticles. But in most cases, when the nanosystem does not have any specific inherent properties that contribute to purification, this step may be a difficult and cumbersome process, and sometimes it is difficult to obtain the purified compound. Common purification techniques useful for various nanosystems are summarized in the table below. Email: info@cd-bioparticles.com Tel: 1-631-633-6938

6. Technique Use Advantages Disadvantages Examples Purification Sterilization Concentration Dispersant changement Reduce size polydispersity Useful for thermolabile compounds Rapid and simple Commercially available devices Cost effective Time consuming Bigger sizes determined by the cut- off size Single-use devices Different sizes of filters Filtration High efficiency Rapid, facile, and economic Low amounts of sample Appropriate for different kinds of nanomaterials Purification Concentration Dispersant changement Special equipment required for large volumes Dificult to resuspend soft matter Conventional centrifugation Ultracentrifugation Gradient centrifugation Centrifugation Conventional or passive dialysis Donnan dialysis Electrodialysis Microdialysis PAGE electrophoresis Electrophoretic mobility shift assay Purification Concentration Dispersant changement Rapid, facile, and economic Commercially available devices No sample pretreatment Limited to the membranes Molecular Weight Cut Off High receptor solution volumes Dialysis Simple and economic High resolution and sensitivity Postelectrophoresis purification steps Need of charged compounds Electrophoresis Purification References: 1. Fornaguera, C., & Solans, C. (2018). Analytical Methods to Characterize and Purify Polymeric Nanoparticles. 2. Han, J., Zhao, D., Li, D., Wang, X., Jin, Z., & Zhao, K. (2018). Polymer-based nanomaterials and applications for vaccines and drugs. , 10(1), 31. , 2018. International Journal of Polymer Science Polymers For more information, view our website: www.cd-bioparticles.com Email: info@cd-bioparticles.com Tel: 1-631-633-6938 Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Fax: 1-631-938-8221 5