How to Characterize and Purify Your Polymer Nanoparticles

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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 speci?c sites. The biomedical
?eld 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. Classi?cation, application areas,
advantages, and disadvantages of polymer-based
nanomaterials
Classi?cation
Materials
Application Areas
Advantages
Disadvantages
Chitosan
Hemostasis material, medical dressing, hydrogel,
drug delivery carrier, gene transfer
Biocompatibility, antimicrobial, innocuous,
easily degradable, adsorbability, ?lm formation
Poor spinnability, poor strength, low
water-solubility
Starch
Hemostasis material, tissue- engineered
scaffold, drug delivery carrier, bone repair
material
Extensive sources, low price, degradation
products safe and non-toxic, non-antigenic
Poor mechanical properties, resistance to water,
poor blocking performance
Natural polymeric material
Alginate
Pharmaceutical excipient, pepcid complete,
medical dressing
Hypotoxicity, biocompatibility, suppresses tumor
growth, enhances immunity
Bad biodegradability, cell attachment poor
Cellulose
Pharmaceutical adjuvant
Extensive sources, low price
Rare adverse reactions
(continued)
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Table 1. (continued)
Biosynthesis material Poly ß- hydroxybutyrate (PHB) Drug-delivery carrier, tissue engineering material Biodegradable, safe, non-toxic, good physical and chemical properties High crystallinity, bad thermal stability
Chemosynthes material (Copolymer) Polylactic (PLA) Anti-adhesion materials, patch, drug-delivery carrier, bone-?xing device, suture, tissue-engineered scaffold Biocompatibility, good mechanical properties, safe, non-toxic Poor toughness, degradation speed slow, hydrophobicity, lack of reactive side chain groups
Chemosynthes material (Copolymer) Polyurethane Excipients, medical bandage Low cost, rich resource, good mechanical properties Degradation speed slow
Chemosynthes material (Copolymer) Poly(lactic-glycolic acid) (PLGA) Absorbable suture, drug delivery, bone screw ?xation, tissue repair Controllable biodegradability, biocompatibility Higher cost, drug-loading capacity and stability can be improved
Chemosynthes material (Copolymer) Polymethyl methacrylate resin (PMMA) Bone-?xation materials, dental materials, arti?cial 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 modi?cation (or lack of
surface modi?cation) has been shown to affect
biocompatibility and increase toxic side effects.
A small change in any of these parameters may
adversely affect the ef?cacy 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.
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• We will brie?y introduce the most common
  analytical techniques used to evaluate these
  parameters, as well as the puri?cation 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 quanti?cation 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 ?lled with a
  solid adsorbent material. The interaction of each
  component in the sample with the adsorbent
  material is slightly different, resulting in
  different ?ow rates for each component and
  separating the components as they ?ow out of the
  column. More interactions between the molecules
  and the column ?lling 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
  ?ltration 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 ?lled 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.

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• 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
  con?rm 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 ?uorescent signal usually
  produced by aromatic molecules to detect and
  characterize organic and inorganic compounds by
  applying a ?uorescent 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 ?uorescent laser and emit ?uorescence 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 ?ow
  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 prede?ned 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.

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Technique Characteristics that analyses Advantages Disadvantages
GPC Molecular weight Rapid and simple High resolution Interaction sample with column ?ling
HPLC Quanti?cation of actives Puri?cation High resolution Rapid and easy performance Low cost/sample Small sample volumes Interaction sample with column ?ling
SEC Puri?cation High resolution Rapid and simple Interaction sample with column ?ling Need of a labelling tag
FTIR Chemical composition Fast and inexpensive Characteristic of each material Possible quanti?cation Complicated sample preparation Interference of water Relatively low sensitivity Requires dried samples Destructive
UV-Vis Quanti?cation of concentration Size and shape determination Cost effective Simple and fast Useful for a variety of compounds Interference between materials
Fluorimetry Quantitative determination ?uorescence High sensitivity Compound speci?city Limited to ?uorescent compounds Limited ?uorescence lifetime
DSC Glass transition Melting temperature Low amounts of sample High precision and sensitivity Requires sample preparation Requires an appropriate reference
TGA Weight loss Low amounts of sample High precision and sensitivity Requires sample preparation Requires an appropriate reference
Puri?cation Techniques In order to ensure that
the formulation is safe and free of contaminants,
it is strongly recommended to carry out the
puri?cation step and then physical and chemical
characterization before starting the preclinical
and clinical analysis of the polymer
nanoparticles. There are various methods for
puri?cation of colloidal nanomaterials, such as
magnetic separation of magnetic nanoparticles.
But in most cases, when the nanosystem does not
have any speci?c inherent properties that
contribute to puri?cation, this step may be a
dif?cult and cumbersome process, and sometimes it
is dif?cult to obtain the puri?ed compound.
Common puri?cation techniques useful for various
nanosystems are summarized in the table below.
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Technique Use Advantages Disadvantages Examples
Filtration Puri?cation 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 ?lters
Centrifugation Puri?cation Concentration Dispersant changement High ef?ciency Rapid, facile, and economic Low amounts of sample Appropriate for different kinds of nanomaterials Special equipment required for large volumes Di?cult to resuspend soft matter Conventional centrifugation Ultracentrifugation Gradient centrifugation
Dialysis Puri?cation 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 Conventional or passive dialysis Donnan dialysis Electrodialysis Microdialysis
Electrophoresis Puri?cation Simple and economic High resolution and sensitivity Postelectrophoresis puri?cation steps Need of charged compounds PAGE electrophoresis Electrophoretic mobility shift assay

• References
• Fornaguera, C., Solans, C. (2018). Analytical
  Methods to Characterize and Purify Polymeric
  Nanoparticles. International Journal of Polymer
  Science , 2018.
• Han, J., Zhao, D., Li, D., Wang, X., Jin, Z.,
  Zhao, K. (2018). Polymer-based nanomaterials and
  applications
• for vaccines and drugs. Polymers , 10(1), 31.

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