Application of Sartorius ultrafiltration products in the preparation of biomedical nanocarriers

Dr. Hannes Landmann, Sartorius Lab Instruments (Göttingen, Germany)

Dr. Kristin Menzel, Scientific Writer (Göttingen, Germany)

In 1908, Paul Ehrlich was inspired by the concept of "Zauberkugel", which for the first time theoretically described the assembly of toxic drugs onto so-called "nanocarriers". 1 Today, nanocarriers have many applications in modern medicine and biotechnology. A key application of these special nanomaterials is directed drug delivery, which acts as a pharmaceutically active transport module (ie, nanoparticles, vesicles, or micelles). 2, 3, 4, 5 It is speculated that this method is more effective and less toxic than the traditional method of administration. 6 In addition to drug delivery, other fields of application of nanocarriers have been developed over the past decades, such as magnetic resonance imaging or stem cell gene therapy using metal nanoparticles, 7 , 8 or optical imaging using quantum dots. 9

Nanocarriers can be classified according to their starting materials (ie, metals, lipids, polymers, and proteins) and post-production compositions (ie, vesicles, particles, and micelles). In general, the preparation of a nanoparticle suspension or vesicle dispersion in an aqueous medium involves three steps: a) nanocarrier assembly (eg by injection, thin film hydration or reverse phase evaporation), b) purification (eg chromatography) , dialysis or ultrafiltration) and c) concentration (eg ultrafiltration or evaporation).

This short review gives an example of some recent literature on the preparation of nanocarriers. It focuses on the concentration and purification step of using a different pore size (corresponding to a molecular weight cutoff (MWCO)) or a Sartorius Vivaspin ® by ultrafiltration device Vivaflow ® completed. The Vivaspin ® range ranges from 0.5 mL to 20 mL, while the Vivaflow ® system covers the range from 0.05 to 5 liters. As a result, Sartorius is able to handle sample volumes, membrane materials and MWCO ranges that are unmatched to meet the needs of different intended applications. Challenges in this area include buffer replacement after synthesis, desalting and cleaning 10,11 , removal of dissolved compounds 12, 13, 14 or aggregates. 15

The purification process is important to achieve an isotonic state through purification, so as to avoid aggregation or agglomeration when applied in vivo, and to remove toxic drugs, ligands or other substances that may cause side effects. The importance of the concentration step is to modulate the amount of active ingredient in the drug to achieve the desired therapeutic or diagnostic effect.

Upon purification, the free material (starting material) is separated from the expected nanocarrier by size exclusion chromatography (SEC), which inevitably leads to product dilution and requires a subsequent concentration step. In contrast, diafiltration filtration does not result in significant dilution, but if a higher nanocarrier concentration is required, a concentration step is still required. Both separation methods require a large amount of expensive and time consuming manual operations. This disadvantage can be overcome by using Vivaspin ® centrifugal or peristaltic pump Vivaflow ® ultrafiltration system. The technology is low in cost, fast in operation, and has few manual operations. It is worth mentioning that the purification and concentration steps can be carried out simultaneously. 16

After purification of the nanocarrier, it is usually necessary to determine the drug loading (coupling or encapsulation efficiency). Coupling or encapsulation efficiency is a reference for the description and identification of nanocarriers. Other important properties include zeta potential and particle size distribution, which can be determined by photon correlation spectroscopy (PCS), high resolution transfer electron microscopy (HRTEM) imaging, or dynamic light scattering (DLS). The suspension or dispersion needs to be successfully purified and concentrated prior to performing these various assays.

The following table summarizes the literature on the purification and concentration of various nanocarriers using the ultrafiltration step. The table also guides you to how large the MWCO is.

Table 1 summarizes the application example using Sartorius Vivaspin ® or Vivaflow ® ultrafiltration nano carrier:

Nanocarriers: nanoparticles, vesicles, micelles

The particle size distribution is obtained by (HR) TEM or DLS , and the Z- average is obtained by PCS or other methods ( if reported )

application

Nanoparticles from metals, metal oxides and functionalized metals

Iron oxide nanoparticles with a cisplatin-containing polymer coating

SD: 4.5 ± 0.9 nm (X-ray diffraction analysis)

Magnetic resonance imaging

Functionalized iron oxide nanoparticles

SD: 38 and 40 nm (DLS method)

Stem cell gene therapy and tracking

Gold nanoparticles

SD: 0.8 – 10.4 nm (atomic force microscopy)

Antibacterial activity

Protein coated gold nanoparticles

SD: 15 and 80 nm (TEM method)

Drug delivery

Functionalized gold nanoparticles

Core SD: 2 nm (TEM method)

Targeted imaging tools and antigen delivery

Functionalized ruthenium nanoparticles

Z-average: 1.1 ± 0.6 nm and 4 – 14 nm

Diagnostic and therapeutic applications

Functionalized nanocrystal

SD: 10 to 20 nm

Quantum dot imaging

Nanoparticles from polymers, functionalized polymers and polymer vesicles

Polymer nanoparticles

Drug delivery

Gelatin coated polymer nanoparticles

Z-average: 280 – 480 nm, depending on composition

Macrophage stimulating activity and drug delivery

Docetaxel-carboxymethylcellulose polymer nanoparticles

Z-average: 118 ± 1.8 nm

Anticancer efficacy study

Functionalized polymer vesicle

Z-average: 185 nm

Surface functionalization research

Lipid nanoparticles and liposomes

Liposomes and micelles

Z-average: liposome 100 nm, micelle 15 nm

Ischemia reperfusion injury

Solid lipid nanoparticles

Z-average: 100 – 120 nm, depending on the lipid used

Drug delivery (brain targeting)

Bacterial outer membrane vesicle

SD: 124 nm (TRPS method)

Adjustable Resistance Pulse Sensing (TRPS) Analysis

Bacterial outer membrane vesicle

basic research

Bacterial outer membrane vesicle

SD: 95 nm

basic research

Bacterial outer membrane vesicle

SD: 50 – 150 nm (TEM method)

basic research

Liposomes

Drug delivery

Liposomes

Capsule hydrophilic drug

(drug delivery)

Micellar

Micellar

Drug delivery

Polymer-based hydrophobic drug micelle

SD (DLS method): 39 – 165 nm, depending on the compound used

Drug delivery

Protein nanoparticle

Protein nanoparticle

SD: 20 – 40 nm (DLS method)

Drug carrier research

SD = particle size distribution

Sartorius ultrafiltration

equipment

MWCO

Ultrafiltration purpose

reference

literature

Vivaspin ® 20

100 kDa

Purification and concentration steps

7

Vivaspin ® 20

100 kDa

Cleaning step

8

Vivaspin ® 20

5 kDa

Purification step

17

Vivaspin ® 6

10 kDa

Separating nanoparticles | Dyes and cleaning

18

Vivaspin ®

10 kDa

Purification step

19

Vivaspin ®

5 kDa, 10 kDa

Purification and concentration

20, 21

Vivaspin ®

300 kDa and

50 kDa

(Before counting) Separation of quantum dot-antibody conjugates from the starting material

9

Vivaspin ®

30 kDa

Purification and concentration

twenty two

Vivaspin ® 20

3 kDa

Cleaning

twenty three

Vivaspin ®

10 kDa

Concentration step

twenty four

Vivaspin ® 20

10 kDa

Concentration step

3

Vivaspin ® 20

100 kDa

Concentration step

25

Vivaflow ® 50

100 kDa

Purification step

26

Vivaflow ® 200

100 kDa

Buffer replacement and concentration steps

27

Vivaspin ® 20 and 500

100 kDa

Buffer replacement and concentration steps

28

Vivaflow ® 200

100 kDa

Buffer replacement and concentration steps

29

Vivaspin ®

100 kDa

Buffer replacement and concentration steps

30

Vivaspin ®

100 kDa

External buffer replacement

2

Vivaflow ® 50

100 kDa

Remove free drug

31

Vivaspin ®

30 kDa

Separation of free material and concentration steps

4

Vivaflow ®

Surfactant removal

14

Vivaspin ® 500

3 kDa

Separation of free drug from capsule drug (quantitative drug binding quantification by subsequent UV-visible analysis)

32

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Paul Ehrlich's magic bullet concept: 100 years of progress8, 473-480 (2008).

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Liposomal doxorubicin for active targeting: Surface modification of the nanocarrier evaluated in vitro and in vivochallenges and prospects Oncotarget 6, (2015).

3. Klermund, L, Poschenrieder, S &t Castiglione, K:

Simple surface functionalization of polymersomes using non-antibacterial peptide anchors. J. Nanobiotechnology 14, 48 (2016).

4.Mulder, WJM et al..

Molecular imaging of macrophages in atherosclerotic plaques using bimodal PEG-micelles.

Magn. Reson. Med. 58,11641170 (2007).

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Nanoparticles in modern medicine: state of the art and futurechallenges.

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Development and characterization of magnetic iron oxide nanoparticles with a cisplatin-bearing polymer coating for targeted drug delivery. IntJ Nanomedicine93659- 3676 (2014).

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Multi-modal transfection agent based on monodispersemagnetic nanoparticles for stem cell gene delivery and tracking. Biomaterials 35, 72397247 (2014).

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Use of quantum dot luminescent probes to achieve single cell resolution of human oral bacteria in biofilms. Appl. Environ. Microbiol. 73, 630- 636 (2007).

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New insights into the structure of PAMAM dendrimer/goldnanoparticle nanocomposites. Langmuir 27, 6759-6767 (2011).

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14 Zhang, Y. etal: Therapeutic surfactant-stripped frozen micelles. Nat Commun, 11649 (2016).

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16.Simonoska Crcarevska, M. et al: Definition of formulation design space, in vitro bioactivity and in vivo biodistribution for hydrophilic drug loaded PLGA/PEOPPO-PEO nanoparticles using OFAT experiments.Eur.. J. Pharm. Sei. 49, 65- 80 (2013).

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21. Faure, ACetal Control of the in vivo biodistribution of hybrid nanoparticles with different poly(ethylene glycol) coatings. Small5, 2565-2575 (2009).

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Curdlan-conjugated PLGA nanoparticles possess macrophagestimulant activity and drug delivery capabilities. Pharm. Res.32.27132726 (2015).

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Preclinical pharmacokinetic, biodistribution and anticancerefficacy studies of a docetaxel-carboxymethycellunanoparticle in mouse mooels. Biomaterials 33,14451454 (2012).

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Passive targeting of lipid-based nanoparticles to mousecardiac ischemia-reperfusion injury. Contrast Media Mol.Imaging8, 117-126 (2013).

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Brain-targeted delivery of resveratrol using solid lipid nanoparticles with functionalized with apolipoproteinEJNanobiotechnology 14, 27 (2016).

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Analysis of bacteria derived outer membrane vesicles usingtunable resistive pulse sensing. Prog. Biomed. Opt. Imaging =Proc. SPIE 9338, 4-9 (2015)

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