Flow field-flow fractionation and multi-angle light scattering as a powerful tool for the characterization and stability evaluation of drug-loaded metal–organic framework nanoparticles

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RESEARCH PAPER

Flow field-flow fractionation and multi-angle light scattering

as a powerful tool for the characterization and stability evaluation

of drug-loaded metal

–organic framework nanoparticles

Barbara Roda

1,2

&

Valentina Marassi

Andrea Zattoni

1,2

Francesco Borghi

Resmi Anand

Valentina Agostoni

Ruxandra Gref

Pierluigi Reschiglian

1,2

Sandra Monti

Received: 15 March 2018 / Revised: 22 May 2018 / Accepted: 29 May 2018

# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and

refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal

–organic frameworks)

loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded

with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native

drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and

the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements

include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their

interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug

delivery applications and gave fundamental data on their size distribution and stability.

Keywords Flow field-flow fractionation . Multi-angle light scattering . Metal

–organic frameworks . Azidothymidine . NRTI .

Metal

–organic framework nanoparticles characterization

Introduction

One of the most used strategies in HIV therapy is the inhibi-

tion of retrotranscription and synthesis of proviral DNA. The

class of drugs which exert such a function are the nucleoside

reverse transcriptase inhibitors (NRTIs), among which azido-

thymidine (AZT) is the most used. It has been demonstrated

that NRTIs exert their antiretroviral activity via metabolization

into their triphosphate derivatives [

]; however, the process,

which is actuated by intracellular kinases, has very low

efficiency [

]. On the other hand, the direct administration

of active triphosphorylated derivatives has important clini-

cal drawbacks, because the high hydrophilicity and poor

stability of such drugs in biological media severely limit

cellular uptake and therapeutic efficiency [

–

4

]. To over-

come this bottleneck, alternative strategies of administra-

tion of NRTIs, based on nanostructured carriers able to

improve drug stability, have been developed to optimize

delivery inside the cells and decrease toxicity effects

[

–

]. In this context, metal

–organic frameworks

(MOFs) have gained increasing attention in the recent

years. MOFs are hybrid crystals composed of metal ions

or clusters connected by multidentate organic ligands and

form structures with very high porosity and very large

surface area. Such unique features increased their interest

for a variety of scientific and technological areas, from

magnetic and electrooptical devices, gas storage, gas/

vapor separation, catalysis, and sensing [

]. In the bio-

logical field, they exhibited a promising potential for im-

aging and drug delivery [

,

17

]. In particular, they have

been proposed as valuable non-toxic nanocarriers for

* Barbara Roda

barbara.roda@unibo.it

Department of Chemistry

BG.Ciamician^, University of Bologna,

Via Selmi 2, 40126 Bologna, Italy

byFlow srl, Via Caduti della Via Fani, 11/B, 40127 Bologna, Italy

CNR-Istituto per la Sintesi Organica e la Fotoreattività, Via Piero

Gobetti, 40129 Bologna, Italy

Institut des Sciences Moléculaires d

’Orsay, UMR CNRS 8214,

Paris-Sud University, Paris Saclay, 91400 Orsay, France

Analytical and Bioanalytical Chemistry

https://doi.org/10.1007/s00216-018-1176-6

several anticancer and antiviral drugs, like busulfan [

–

], cidofovir [

], doxorubicin [

,

21

], and

NRTIs like AZT-TP [

] and gemcitabine [

–

Nanoscale iron trimesate metal

–organic frameworks (MIL-

100(Fe) nanoMOFs, MIL = Materials of Institut Lavoisier)

exhibit a biodegradable and biocompatible crystalline struc-

ture with high and regular porosity, formed by the spontane-

ous formation of iron(III) octahedral trimers in the presence of

trimesic acid [

,

25

]. The resulting 3D mesoporous sys-

tem presents smaller cages (24 Å) accessible through pentag-

onal microporous windows (5.6 Å) and larger cages (29 Å)

delimited by both pentagonal and hexagonal apertures (8.6 Å).

MIL-100(Fe) nanoMOFs showed very high AZT-TP loading

capability (up to 24 wt%) together with controlled release

abilities [

,

22

]. Due to the small size and flexible structure,

AZT-TP molecules are most likely adsorbed within the larger

mesoporous cages and interact through the phosphate groups

with the unsaturated iron(III) Lewis acid sites. This strong

binding further ensures a sustained in vitro delivery of the

AZT-TP ligand within HIV-infected cells, inhibiting up to

the 90% of the viral replication [

]. Such encouraging

perspectives have been further confirmed by an investigation

of the encapsulation process in the AZT-monophosphorylated

derivative (AZT-MP). Understanding of the impact of the

number of phosphate groups per nucleoside molecule on the

drug-nanocarrier association has direct relevance to the poten-

tial use of NRTIs monophosphorylated form as prodrug. It is

generally considered that the first phosphorylation step is the

rate-limiting reaction in the metabolic activation of nucleoside

analogues [

As nanostructured materials for biological applications,

nanoMOFs have to meet stability requirements. A certain de-

gree of chemical instability in the material is a desirable prop-

erty in physiological conditions, since the nanocarrier is expect-

ed to deliver and release the drug into the cell and once this

function is completed it should be able to degrade in situ. On

the other hand, for preparation and characterization purposes, a

good stability of the colloidal suspensions is required. For un-

derstanding the factors that govern the stability of the nanopar-

ticles (NPs) upon drug encapsulation and release, the charac-

terization of the suspensions as regards particle size and mor-

phology is crucial [

]. Such study is usually carried out by

techniques like atomic force microscopy and scanning or trans-

mission electron microscopy, and spectroscopic techniques like

dynamic light scattering (DLS) [

]. Electron microscopy

allows direct observation of the individual particles and allows

to assess their shape and size, but time-consuming analyses

have to be performed to provide significant data from a statis-

tical point of view, while DLS allows for the measurement of

average dimension over the entire population of particles but

does not provide information about morphology.

Field-flow fractionation is a family of separation tech-

niques suitable for the characterization of nanosized and

microsized systems. The separation is obtained as a combina-

tion of a parabolic flow of carrier solution and an external

field. Among the various sub-techniques, asymmetric flow

field-flow fractionation (AF4) is the one which experienced

the greatest success and development and is in fact the most

established and commercially available [

–

31

]. The separa-

tion results from the application of a flow stream of carrier

along a channel with a capillary dimension together with a

perpendicular, hydrodynamic flow. Since the separation de-

pends only upon the carrier flows applied and because of the

lack of a packed stationary phase, AF4 has a unique gentle

separation mechanism: issues like shear forces or particle al-

teration are therefore avoided. Retention times of particles are

inversely proportional to their diffusion coefficient (D), and

directly proportional to the hydrodynamic diameter [

]. The

two perpendicular flows are generated by splitting the longi-

tudinal (pump delivered) flow into a second one (namely the

cross-flow or x-flow) across the accumulation wall (the only

permeable wall of the channel).

Multi-angle light scattering (MALS) detection is an abso-

lute method for particle sizing and molar mass determination

and is able to work over a wide dimensional range without the

use of any standard [

]. The theory on size and mass calcu-

lation based on static light scattering had been extensively

explained [

]. Briefly, when refractive index increment (dn/

dc) and sample concentration are known, MALS can provide

the molecular weight (Mw), while, independently of concen-

tration or other sample specification and without assumptions

on particle conformation or shape, it provides the root mean

square (rms) radius, which represents the mass-averaged dis-

tance of each mass element of the NP from its center of grav-

ity. If both Mw and rms radius distributions are known, parti-

cle shape and density information are accessible (deriving

from the Mw-to-rms ratio). MALS detection is applicable in

the range 10 nm

–1 μm.

The literature reports that AF4-MALS combination has

been successfully used for size analysis, study of stability,

and drug release of NPs of different composition, since it

allows for accurate size distribution determination, investiga-

tion of NPs aggregation in native conditions, separation of the

unbound constituents of the functional NPs, and determina-

tion of the optical features of the NPs (separated from other

dispersion components including free chromophores, or free

drugs) [

–

]. Some examples of use of AF4-MALS com-

bination on drug delivery NPs are relevant to the characteri-

zation of lipidic/liposome suspensions [

–

], organic poly-

mer particles like micelles (amphiphilic molecules),

polymerosomes, dendrimers, nanocapsules, and polymeric

NPs [

42

–

] and biopolymer particles [

–

52

In this work, we propose application of AF4 coupled with

MALS detection for the characterization of MIL-100(Fe)

nanoMOFs loaded with NRTI drugs for the first time. The

goal is to determine (a) the particle size distribution (PSD),

Roda B. et al.

(b) the stability of MOF NPs, and (c) the particle interaction

with the drug in a time frame of 24 h. To this aim, four types of

samples were examined: empty nanoMOFs and nanoMOFs

loaded with AZT derivatives with three different degrees of

phosphorylation: azidothymidine (AZT, native drug), azido-

thymidine monophosphate (AZT-MP), and azidothymidine

triphosphate (AZT-TP). It was also possible to relate the

MOF NP stability to the nature of the interaction with the drug

molecules.

Experimental part

Materials

Iron(III) chloride hexahydrate (Alfa Aesar, 98%) and 1,3,5-

benzenetricarboxylic acid (1,3,5-BTC; Aldrich, 95%) were

used as reactants for the nanoMOF synthesis and absolute

ethanol (Carlo Erba, 99%) for their activation. 3

′-azido-3′-

deoxythymidine (AZT, azido 3

′-deoxythymidine, Moravek),

′-azidothymidine monophosphate (AZT-MP, 3′-azido-3′-de-

oxy-

-thymidine 5

′-monophosphate sodium salt, Carbosinth),

and 3

′-azidothymidine triphosphate (AZT-TP, 3′-azido-2′,3′-

dideoxythymidine-5

′-triphosphate lithium salt, TriLink) were

used as received. Water was purified by passage through a

Millipore MilliQ system (Millipore SpA, Milan, Italy).

Water was used as dispersing medium.

Synthesis of the MIL-100(Fe) nanoMOFs was achieved by

means of a hydrothermal microwave-assisted reaction de-

scribed elsewhere [

], starting from a mixture of iron

chloride (8.97 mmol) and 1,3,5-benzenetricarboxylic acid

(4.02 mmol) in 20 ml of deionized water, heating 6 min at

130 °C under stirring. The as-synthesized nanoMOFs were

recovered by centrifugation (10 min, 10,000 g). To remove

the residual non-reacted organic acid, they were washed in

50 ml of absolute ethanol and recovered by centrifugation

(10 min, 10,000g). This activation step was repeated six times.

MIL-100(Fe) nanoMOF characterization was performed

by means of several techniques. Crystallinity was assessed

by X-ray powder diffraction (XRPD); NP diameter and mor-

phology were determined by dynamic light scattering (DLS)

and transmission electron microscopy (TEM). The porous sur-

face was measured by nitrogen adsorption analysis at

−

196 °C. The calculated BET surface was 1650 ± 100 m

−1

For any detail about application of the mentioned techniques,

see previous publications [

Samples

MIL-100(Fe) nanoMOFs were stored in the dark at room tem-

perature as EtOH wet material. An aliquot was suspended in a

few milliliters of ethanol, then centrifuged (10 min, 10,000g)

and subsequently washed two times with water to completely

remove ethanol. Four samples were prepared, one was empty

nanoMOFs and the other three were drug-loaded nanoMOFs.

Aliquots of the centrifugate were taken and re-dispersed in

water or aqueous solution of drug to achieve a final concen-

tration of 0.2 mg/ml of the starting solid MOF material and

0.2 mg/ml of MOFs and 0.1 mM of drug for the drug-

conjugated particles. NanoMOFs were loaded with drugs sim-

ply by impregnation. Nanoparticles were incubated with the

aqueous solution of drug, stirring the mixture at room temper-

ature in the dark for either 15 min (freshly prepared samples)

or 24 h. With freshly prepared samples, all the measurements

were performed within 1 h from preparation. The 24-h delay

was chosen to evaluate the stability of the MOF NP conjugat-

ed with the phosphorylated AZT derivatives, because no sig-

nificant release of the drugs was assessed by HPLC analysis

over 3 days in water [

].

Measured pH value of dispersed MOF NPs was slightly

acidic.

The zeta potential (ZP) of NPs was measured with a

Zetasizer 4 equipment (Malvern Instruments, UK) on suspen-

sions containing 0.2 mg/mL of MOF NPs.

AF4-MALS analysis

AF4-MALS was performed by using a 1100 Series HPLC

system (Agilent Technologies, Palo Alto, CA), connected to

a control module to control AF4 flow rates and operations

(Eclipse 3, Wyatt Technology Europe, Dernbach, Germany).

On-line detection of the eluted species was performed with an

Agilent 1100 DAD UV/Vis spectrophotometer, a MALS

DAWN HELEOS detector (Wyatt Technology Corporation,

Santa Barbara, CA), and an Optilab rEX refractive index de-

tector (Wyatt Technology Corporation). Carrier solutions were

degassed using an on-line vacuum degasser Agilent, 1100

series (Agilent Technologies).

The separation device is constituted by a flat channel with a

trapezoidal shape and capillary height. For the sample analy-

sis, particles are introduced in the channel and focused,

allowing the sample to concentrate on a narrow band. When

the elution starts, the separation is gained by the combination

of a longitudinal and a perpendicular hydrodynamic field. The

channel was 152 mm long (Wyatt Technology Europe),

equipped with a polyethersulfone membrane (Nadir), with a

molecular weight cutoff of 10 kDa. The channel spacer was

350

μm thick, with trapezoidal shape (upstream width b0 =

16 mm; downstream width bL = 4 mm).

The flow rate program was set up as follows: 2 min of

elution (crossflow 0.2 ml/min) was applied to equilibrate the

baselines on the detectors; then 1-min focus flow (0.5 ml/min)

was applied to equilibrate the flows in the channel. Then 2-

min injection (at a flow rate of 0.2 ml/min) in focus mode was

applied in order to allow the sample to reach the channel, and

2 further minutes of focus was used to allow for a complete

Flow field-flow fractionation and multi-angle light scattering as a powerful tool for the characterization...

relaxation. After the focus step, the elution starts with an

isocratic cross-flow step of 0.2 ml/min for 30 min. Elution

medium was pure water. The method reproducibility was ver-

ified in the method development step to ensure robustness and

the effective correspondence between retention time and sam-

ple properties depending on time and storage conditions.

Results and discussion

AF4-MALS characterization of empty nanoMOFs

MIL-100(Fe) nanoMOFs were successfully prepared and

characterized as described in

Materials

^ section. DLS and

TEM showed particle diameters around 200

–240 nm and

facetted-type architecture [

,

22

Figure

reports the light scattering signal and the rms radius

distribution obtained by the AF4-MALS analysis of empty

nanoMOF particles in the freshly prepared sample (measure-

ment performed within 1 h from preparation). The fractionation

profiles constituted by a monomodal but relatively large distri-

bution band, with a maximum at retention time (tR) of 18 min

and a tail at higher retention times (up until 22

–23 min). The

particle size distribution (PSD) obtained by the AF4-MALS

fractionation indicates that the sample is fairly homogenous

with rms radius values of ca. 81 nm (see Table

).

As for the fractionation method, the absence of peak at the

field release (tR = 37 min) indicates a complete fractionation

process with no retained sample into the channel. In addition,

the profile has a sharp peak at tR = 8 min which is the void

peak corresponding to the flow change effect together with a

negligible part of non-retained sample; this is a constant for all

injections.

Comparison of the AF4-MALS rms radius value with di-

ameter obtained from DLS (200

–250 nm, see ref. [

]) indi-

cates a fair agreement. More in detail, the parameter that

MALS measures is the root mean square (rms) radius, the

measure of a particle

’s size weighted by the mass distribution

about its center of mass, while DLS and nanoparticle tracking

analysis (NTA) give a measure of the hydrodynamic radius

(Rh). The ratio between rms and Rh (shape factor) gives in-

sight into the actual shape of the particles. For example, a ratio

of 1.7 (rms/Rh) corresponds to rods or chain aggregates, while

a ratio of about 0.7

–0.8 corresponds to more compact spher-

ical objects [

]. NanoMOFs show a MALS-calculated diam-

eter and a DLS-calculated diameter of 162 and 210 nm respec-

tively; the ratio would be 0.77. This is in line with a globular

compact mesoporous material, in agreement with TEM and

XRDP findings [

] indicating a facetted and crystalline struc-

ture, and with N2 absorption findings showing an important

porous structure [

].

By correlating the characterization data with the AF4-

MALS results, we can conclude that the AF4-MALS system

appears suitable for the characterization of nanoMOF suspen-

sions and can allow to obtain fundamental data on their size

and stability over time as it could detect even minor size

evolutions.

AF4-MALS characterization of drug-loaded nanoMOFs

The PSD in the nanoMOFs loaded with AZT, AZT-MP, and

AZT-TP was measured in the freshly prepared suspensions

and in the suspensions stored 24 h in the dark at room tem-

perature with the developed AF4-MALS method used for

empty nanoMOFs. In Fig.

, the fractographic profiles of the

three freshly prepared, drug-loaded nanoMOF suspensions

Fig. 1 Fractograms (LS signal at

90°) and calculated gyration radii

of empty nanoMOF; freshly

prepared sample: 0.2 mg/mL of

solid MOF material, in water

Table 1 rms radius

values (nm) of

nanoMOFs immediately

after preparation and af-

ter 24 h. All samples

were at a concentration

of 0.2 mg/mL of

nanoMOF in water

Sample

rms radius (nm)

Fresh

24 h

Empty nanoMOF

81 ± 2

–

NanoMOF-AZT

81 ± 2

80 ± 3

NanoMOF-AZT-MP

90 ± 2

NanoMOF-AZT-TP

97 ± 2

Roda B. et al.

(nanoMOF 0.2 mg/mL + AZT 1 × 10

−4

M, nanoMOF 0.2 mg/

mL + AZT-MP 1 × 10

−4

M, nanoMOF 0.2 mg/mL + AZT-TP

1 × 10

−4

M) were compared with those of the corresponding

empty nanoMOF (0.2 mg/mL) suspension. Results of PSD

analysis by AF4-MALS are reported in Table

.

It can be noted that empty nanoMOF and MOF-AZT sam-

ples have identical PSD. The MOF-AZT-MP NP exhibits a

rms radius of 90.0 ± 2.0 nm with an increase of ca. 10 nm with

respect to the empty nanoMOFs. This fact can be rationalized

with the prompt binding of the drug to the nanoMOFs and to

bridging effects leading to a slight increase of the size. The

interaction of the triphosphorylated drug with the MOF NP

frame is also established promptly. The MOF-AZT-TP sample

shows a further increase of NP radius with a measured value

of 96.8 ± 2.3 nm. On the contrary, no increase of the

nanoMOF size is observed with AZT.

These findings clearly (i) confirm the prompt binding of

both AZT-MP and AZT-TP to the nanoMOF frame; (ii) are in

agreement with the known lack of appreciable interaction of the

native drug with the nanoMOFs, assessed by means of drug

loading and release studies combined with UV-vis spectrosco-

py, ITC, and molecular modeling [

]; (iii) highlight the key

role of the phosphate groups for effective encapsulation of AZT

derivatives within the MIL-100(Fe) nanoMOF cages, also tes-

tified by the very high maximum loadings observed, as report-

ed in a previous work (36 and 24 wt% for AZT-MP and AZT-

TP, respectively) [

]; and (iv) support the idea of a different

MOF interplay with the mono- or triphosphorylated drug, al-

ready suggested by UV-Vis absorption spectroscopy, circular

dichroism, isothermal titration calorimetry, and molecular

modeling data [

,

24

]. On the basis of these techniques, the

binding mechanism has been assigned to the formation of

strong iono-covalent links of the phosphate groups with the

Lewis iron(III) acid sites [

]. Due to the stronger complexing

capability, the triphosphorylated drug was proposed to possess

higher tendency than AZT-MP to occupy the nanoMOF cages

by replacing coordinated trimesic acid bound to iron metal

sites. With this respect, it has been also shown that the number

of phosphate groups has a direct impact on the binding constant

which is lower for AZT-MP than for AZT-TP (K = 1340 vs.

2930 M

−1

). These findings are also in agreement with the pre-

viously reported data from XRDP measurements, which shows

that the encapsulation of the phosphorylated drug into the

nanoMOF structure does not affect the crystalline structure

[

], and is performed through pore filling.

The fractograms in Fig.

also indicate that, despite FFF

theory suggesting that smaller particles elute first, the reten-

tion times of the loaded vs. unloaded MOF NPs are inverted:

MOF-AZT-MP and MOF-AZT-TP particles have lower reten-

tion time than empty nanoMOF and MOF-AZT particles. This

reflects a change in the zeta potential of the MOF NPs. Zeta

potential plays indeed a key role in the retention time of the

nanoparticles because it influences the elution process inside

the channel. In Table

, the measured values of the zeta po-

tential for the MOF-AZT-MP and MOF-AZT-TP are com-

pared to those of empty nanoMOFs (AZT-loaded particles

exhibit a surface charge quite similar to that of the empty

ones). The loaded MOF NPs show negative surface charge

with absolute values that are higher when compared to those

of empty ones. Since the channel membrane is negatively

charged, negative conjugated particles are repelled away from

the membrane and travel at a higher position compared to the

unconjugated ones: thus, their position corresponds to a

higher flow stream. The repeatability of these results has been

confirmed in more than seven repetitions under same condi-

tions showing a CV% < 5%: this behavior confirms that AZT-

MP and AZT-TP actually bind to the nanoMOF and are pres-

ent in the porous cores as well as in the top layers influencing

the overall particle potential.

Another indirect indication of drug-nanoparticle complex-

ation comes from the relative intensity of the LS signals. The

higher scattering intensity for the MOF-AZT-MP and MOF-

Fig. 2 Fractograms (LS signal at 90°) and calculated gyration radii of

freshly prepared MOF samples. Light gray dotted line: MOF-AZT-TP.

Gray dotted line: MOF-AZT-MP; Black line: MOF-AZT. Gray line: emp-

ty nanoMOF. Samples: 0.2 mg/ml of the solid MOF material for the

empty nanoMOFs and 0.2 mg/ml of the solid material plus 0.1 mM of

drug for the drug-loaded particles, in water

Table 2

Zeta potential of empty nanoMOF, MOF-AZT-MP, and MOF-

AZT-TP NPs, measured in water at 0.2 mg/ml MOF material

concentration

Sample

Zeta potential (mV)

Empty nanoMOF

+ 10 ± 3

NanoMOF-AZT-MP

− 15 ± 2

NanoMOF-AZT-TP

− 40 ± 2

Flow field-flow fractionation and multi-angle light scattering as a powerful tool for the characterization...

AZT-TP with respect to the signal of the unloaded particles is

not simply justified by the increase in PSD of just 10 and

16 nm, respectively, since the size difference is too small.

Actually, the LS signal is proportional to the mass, concentra-

tion, and derivative of the refractive index vs. concentration,

dn/dc [

]. The different scattering power can be then ex-

plained by taking into consideration the last two parameters.

The dn/dc values are expected to be bigger for the loaded

MOF with respect to the unloaded MOF particles, the formers

being more polarizable and therefore able to give a more in-

tense signal. Indeed, opposed to AZT which has practically no

interaction with the nanoMOFs, AZT-MP and AZT-TP fill

completely their large cages as previously reported [

Moreover, the actual concentration of nude and AZT-

nanoMOFs could lower, due to precipitation of macro-aggre-

gates, thereby reducing the scattering intensity. The colloidal

instability of the suspensions is most evident for nude

nanoMOFs, but also noticeable for AZT-nanoMOF (see also

AF4-MALS study of drug-loaded nanoMOF stability

^ sec-

tion), further proving the lack of interaction between the

nanoMOF and a non-phosphorylated drug.

In conclusion, differences in PSD, particles surface charge,

and polarizability between empty particles and drug-loaded par-

ticles give a strong indication of MOF surface and core modi-

fication, and, together with the signal intensity, may contribute

to the elucidation of the stability features of these systems.

AF4-MALS study of drug-loaded nanoMOF stability

The AF4-MALS method was applied to control the morpho-

logical stability of the drug-loaded nanoparticles after 24 h

(Fig.

–c).

The fractograms relevant to the MOF-AZT system are re-

ported in Fig.

a. The profiles indicate that immediately after

Fig. 3 AF4-MALS profiles of

nanoMOF particles immediately

after preparation (gray line) and

after 24 h (black line) incubated

with

a MOF-AZT, b MOF-AZT-

MP, and

c MOF-AZT-TP.

Samples: 0.2 mg/ml of the solid

material plus 0.1 mM of drug for

the drug-loaded particles, in water

Roda B. et al.

preparation there is a Gaussian and symmetric band, which

becomes bimodal and less intense after 24 h. This fact high-

lights how the MOF-AZT nanoparticles tend to aggregate

with elapsing time. The particle size distribution does not

change so much. A very poor interaction of AZT with the

MOF frame is confirmed also after 24 h. However, the big

decrease in LS signal intensity suggests a decrease in particle

concentration in the sample. Most probably, precipitation of

aggregates and sedimentation could explain such effects, as

mentioned above.

Figure

3

b shows the fractograms of the MOF-AZT-MP

system. After 24 h, there is only a little variation in the LS

signal intensity and in the band profile, and the PSD given by

the rms radius does not change (Table

Figure

c shows the fractograms of the MOF-AZT-TP

system. Also in this case, the signal intensity and band

profile do not change much over the 24 h and the rms

radius keeps the same.

From the comparison between PSD distributions in Fig.

a

–c

and from data in Table

, it can be inferred that all the drug-

loaded systems are fairly stable over the time frame of the ob-

servations. AZT does not modify the nude particle scattering

behavior, thus confirming the lack of appreciable interaction,

whereas the phosphorylated derivatives strongly modify the

scattering behavior due to efficient interaction with the

nanoMOF frame. Comparison of the MOF-AZT-MP system

with the MOF-AZT-TP one indicates the latter is more stable.

This fact could be explained with the magnitude of the apparent

binding constant of the drug to the MOF frame, which is higher

for AZT-TP than for AZT-MP and points to a role of the strength

of the interaction controlled by the number of phosphate groups,

in the system stability [

]. However, the changes in rms radius

over 24 h are negligible for both MOF-AZT-MP and MOF-

AZT-TP, so it can be concluded that the interaction with both

phosphorylated drugs was maintained during storage.

Conclusions

AF4-MALS was for the first time applied to the characteriza-

tion of a nanosized MOF material, MIL-100(Fe) nanoMOFs,

loaded with NRTI drugs of interest for anti-HIV therapy. The

method provided important information on size, stability, and

aggregation behavior of the MOF NPs and clearly evidenced

surface/core modification on the drug-loaded nanoMOF.

AF4-MALS was shown here to be a useful methodology to

analyze particles intended for drug delivery applications. The

size separation together with the unique gentle separation

mechanism is crucial to avoid loss of aggregates and to detect

them, allowing for their identification even when they are

present in such a small amount that they do not affect signif-

icantly the particle size distribution (e.g., when the aggrega-

tion process is at its early stage). The online coupling with

MALS detection allowed for particle size distribution deter-

mination and early-stage aggregation detection. Insights were

obtained on drug location which greatly influenced the dn/dc

parameters of the nanoMOFs.

Acknowledgments

This work has been carried out in the frame of the European

ITN network CYCLON (no. 237962).

Compliance with ethical standards

Conflict of interest

Andrea Zattoni, Barbara Roda, and Pierluigi

Reschiglian are associates of the academic spinoff company byFlow Srl

(Bologna, Italy). The company mission includes know-how transfer, de-

velopment, and application of novel technologies and methodologies for

the analysis and characterization of samples of nanobiotechnological in-

terest. Valentina Marassi, Francesco Borghi, Resmi Anand, Valentina

Agostoni, Ruxandra Gref, and Sandra Monti declare no conflicts of

interest.

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Document Outline

Flow...
- Abstract
- Introduction
- Experimental part
  - Materials
  - Samples
  - AF4-MALS analysis
- Results and discussion
  - AF4-MALS characterization of empty nanoMOFs
  - AF4-MALS characterization of drug-loaded nanoMOFs
  - AF4-MALS study of drug-loaded nanoMOF stability
- Conclusions
- Acknowledgments
- References

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