Gemcitabine Lipid Prodrugs: The Key Role of the Lipid Moiety on the Self-Assembly into Nanoparticles

the Self-Assembly into Nanoparticles

Article

Gemcitabine Lipid Prodrugs: The Key Role of the Lipid Moiety on

Eleonore Coppens, Didier Desma ë le, Julie Mougin, Sandrine Tusseau-Nenez, Patrick Couvreur,

and Simona Mura*

Cite This: Bioconjugate Chem. 2021, 32, 782 −793

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sı Supporting Information

ABSTRACT: A small library of amphiphilic prodrugs has been synthesized by conjugation of gemcitabine (Gem) (a hydrophilic

nucleoside analogue) to a series of lipid moieties and investigated for their capacity to spontaneously self-assemble into nanosized

objects by simple nanoprecipitation. Four of these conjugates formed stable nanoparticles (NPs), while with the others, immediate

aggregation occurred, whatever the tested experimental conditions. Whether such capacity could have been predicted based on the

prodrug physicochemical features was a matter of question. Among various parameters, the hydrophilic−lipophilic balance (HLB)

value seemed to hold a predictive character. Indeed, we identiﬁed a threshold value which well correlated with the tendency (or not)

of the synthesized prodrugs to form stable nanoparticles. Such a hypothesis was further conﬁrmed by broadening the analysis to

Gem and other nucleoside prodrugs already described in the literature. We also observed that, in the case of Gem prodrugs, the lipid

moiety aﬀected not only the colloidal properties but also the in vitro anticancer eﬃcacy of the resulting nanoparticles. Overall, this

study provides a useful demonstration of the predictive potential of the HLB value for lipid prodrug NP formulation and highlights

the need of their opportune in vitro screening, as optimal drug loading does not always translate in an eﬃcient biological activity.

INTRODUCTION

gastrointestinal tract, therefore leading to an eﬃcient delivery

to the target cells or tissues. To a certain extent, the nature of

the lipid moiety allows modulation of the fate of the prodrug

upon administration by exploiting speciﬁc lipid metabolism

■

Clinical eﬃcacy of even extremely promising preclinical drug

candidates is often compromised by unfavorable physicochem-

ical properties such as limited aqueous solubility, poor

permeability, short half-life due to instability in biological

16,17

pathways within the body.

administered drugs can be conjugated to triglycerides to either

facilitate selective absorption into the lymphatic circulation for

lymphocytes and lymph nodes targeting or rejoin the

systemic circulation while bypassing the ﬁrst-pass hepatic

Thus, for instance, orally

media and/or rapid elimination. With the aim of overcoming

these challenging issues, prodrug approaches have been largely

investigated, and their relevance is well conﬁrmed by the

observation that prodrugs represent up to 10% of all new drug

molecules approved by the FDA over the 2008−2017 period.

Prodrugs can be obtained by chemical modiﬁcation of the

parent molecule with small moieties (e.g., ionizable groups

metabolism.

Steroid prodrugs hold potential for cancer cell targeting by

taking advantage of the accumulation of steroids, in particular,

cholesterol, in neoplastic tissues.

prodrugs allow drug delivery to tumors because fatty acids are

key nutrients for cancerous tissues.

formulation may be needed to overcome the lack of aqueous

solubility of these lipid prodrugs and to allow their systemic

19,20

which increase aqueous drug solubility), but derivatization

Likewise, fatty-acid-based

−6

4,8−10

with lipids,

peptides, and polymers

has also been

proposed. The latter enables the protection of the drug from

the degradation and/or to achieve a controlled and sustained

6,21,22

Yet, opportune

1−13

drug release.

Whatever the modiﬁcation, it induces a loss

of the drug activity, which is then recovered in vivo after

enzymatic and/or chemical activation of the prodrug.

Among the diﬀerent types of prodrugs, lipid-based ones, in

which the drug is linked to fatty acids, cholesterol derivatives,

12,13

Received: February 2, 2021

Revised: March 17, 2021

Published: April 2, 2021

and phospholipids, are of particular interest. Not only are

these lipids biocompatible, thus limiting insurgence of

undesired toxicity, but they also could facilitate the crossing

of biological barriers such as the blood−brain barrier or the

2021 American Chemical Society

Bioconjugate Chem. 2021, 32, 782−793

Article

Figure 1. Structure of the gemcitabine and of the lipids used for the synthesis of the prodrugs.

8,40

administration in a clinically acceptable solvent. In this context,

a combination with nanoscale carriers has been proposed and

lipid prodrugs have been successfully loaded into lip-

all restrict pharmacological eﬃcacy.

Conjugation to

squalene advantageously modiﬁed the Gem pharmacokinetics,

leading to a better therapeutic index compared to the free drug,

in several experimental tumor models in mice and rats.

Despite these interesting results, whether the squalene was

the only lipid able to combine both spontaneous self-assembly

of the gemcitabine prodrugs and biological activity was still a

matter of question. We already investigated the inﬂuence of the

isoprene chain length, revealing that at least four isoprene units

were required to formulate prodrug nanoparticles without

3−27

28−31

32−34

41−44

osomes,

polymer nanoparticles (NPs).

solid lipid nanoparticles,

micelles,

5−37

On the contrary to the above-mentioned strategies, in which

the prodrug was encapsulated into other nanocarriers, we have

taken advantage of the dynamically folded conformation of

squalene, a biocompatible triterpene, for drug conjugation and

spontaneous supramolecular assembly as nanoparticles. In

the latter, the drug−squalene conjugate was the only

component of the formed nanoparticles, thus avoiding the

use of additional materials (polymers, lipids, etc.) and

excipients (surfactants, sugars, etc.). This resulted in a

considerable increase of the drug loading, and thanks to a

simpliﬁed composition, it could eventually facilitate scalability

and introduction on the market. Thus, the linkage of squalene

to various drugs resulted in nanoparticles of about 100 nm

diameter and represented a versatile nanomedicine plat-

additional excipients.

In the current study, we extended the nature of the

investigated lipid molecules and we included in our pool (i)

the cholesterol, (ii) the α-tocopherol, a fat-soluble vitamin,

(iii) a few fatty acids, and (iv) a diglyceride. We assessed the X-

ray diﬀraction patterns of the synthesized Gem prodrugs, and

we evaluated their capacity to self-assemble as nanoparticles in

pure water at the minimum concentration of 1 mg·mL ,

necessary for further biological studies. When they were

obtained, the resulting nanoparticles underwent a detailed

physicochemical characterization before being investigated for

their in vitro cytotoxicity on the MDA-MB-231 breast cancer

cell line. The hydrophilic−lipophilic balance (HLB) of the

prodrug library was used to assess whether self-assembly failure

−

8,39

form.

This approach has been ﬁrst applied to gemcitabine (Gem)

with the aim to overcome the major limitations of this

anticancer nucleoside analogue (e.g., short half-life, poor

diﬀusion into cells, and induction of drug resistance), which

Bioconjugate Chem. 2021, 32, 782−793

Bioconjugate Chemistry

Article

Table 1. Formulation Conditions for the Library of Gemcitabine Lipid Conjugates and Resulting Nanoparticle

Characterization

mean

zeta- _a

potential

(mV)

drug

nanoprecipitation

organic solvent

organic solvent/ NP concentration

diame-

polydispersity

conjugate

water ratio

(mg·mL⁻

)

ter (nm)

index

(wt %)

HLB

SQGem

RSQGem

EtOH

THF

EtOH

THF

EtOH

THF

EtOH

1:2

1:4

1:2

89 ± 8

121 ± 3

157 ± 32

112 ± 15

0.09 ± 0.03

0.03 ± 0.02

0.07 ± 0.02

0.23 ± 0.02

0.11 ± 0.03

−25 ± 6

−11 ± 1

−53 ± 8

−31 ± 6

−36 ± 17

40.7

40.1

26.6

35.2

33.3

28.4

49.9

7.41

7.30

6.00

7.95

8.49

8.89

9.07

SolgluGem

CholgluGem

VitEgluGem

DiglygluGem

OAGem

146 ± 17

aggregation

#08

#09

#10

#11

LAGem

50.1

48.1

45.9

39.3

9.10

8.74

8.34

7.14

EPAGem

DHAGem

C28Gem

THF

EtOH

THF

Measured by dynamic light scattering (values ± standard deviation, n ≥ 3). Calculated as molecular weight (gemcitabine)/molecular weight

conjugate). Calculated according to the Griﬃn formula using the MarvinSketch version 21.3 software.

(

of some prodrugs into nanoparticles could have been predicted

ahead of the time-consuming chemical synthesis and

physicochemical evaluation.

RESULTS AND DISCUSSION

■

Synthesis of the Lipid−Gemcitabine Bioconjugates.

Using as a reference the squalene moiety (C H )

characterized by six double bonds symmetrically disposed

around the C −C linkage, a series of lipids displaying

diﬀerent chemical features was chosen to synthesize a library of

gemcitabine prodrugs (Figure 1). Thus, within the terpene

family, the gemcitabine was conjugated to the perhydrogenated

SQ (RSQ) devoid of unsaturation, as well as to the solanesol

(

Sol), a long-chain polyisoprenoid alcohol incorporating nine

isoprene units. Then, polycyclic structures were introduced by

conjugation to cholesterol (Chol) and α-tocopherol (VitE),

which possess a sterol and a chromanol carbon skeleton,

respectively. Prodrugs derived from nonterpenoid lipids

without lateral methyl groups were synthesized by gemcitabine

linkage to fatty acids, both saturated (i.e., octacosanoic acid,

C28) and unsaturated (i.e., oleic (OA), linoleic (LA),

eicosapentaeneoic (EPA), and docosahexaenoic acid

(

DHA)). Last, by derivatization with the glyceryl 1,3-

dipalmitate (Digly), a gemcitabine prodrug with two side

chains was obtained.

As previously reported for the squalene−gemcitabine

prodrug (SQGem, Table 1, #01), the diﬀerent derivatives

were synthesized by covalent conjugation of the lipid moieties

onto the 4′-amino group of the cytosine nucleus of the drug, as

depicted in Figures 2−4. Conjugation on this position allowed

the rapid drug degradation into the inactive diﬂuorodeoxyur-

idine metabolite by action of the endogenous deaminases to be

prevented. For the sake of comparison, aiming to have

conjugates in which the lipid is anchored to the drug via an

amide bond, the synthetic strategies have been opportunely

Figure 2. Synthetic strategy implemented to obtain the lipid prodrugs

when using lipid moieties bearing a carboxylic group without

protection of the gemcitabine moiety.

adapted according to the functional group available on the

former. When necessary, a glutaryl spacer was introduced

between the lipid moiety and the amino group. Hence, lipids

bearing a carboxylic acid function were ﬁrst activated into the

Bioconjugate Chem. 2021, 32, 782−793

Figure 3. Synthetic strategy applied to obtain the C28Gem lipid prodrug.

Article

Figure 4. Synthetic strategy applied to obtain the lipid prodrugs while using lipid moieties bearing an OH group.

mixed anhydride using ethyl chloroformate and then reacted

with the gemcitabine base (Figure 2) or its bis-3′,5′-tert-

butyldimethylsilyl-protected form (Figure 3). The release of

gemcitabine from these prodrugs is expected to occur in vivo

after proteolytic cleavage of the amide bond by the lysosomal

cathepsins (B and D), as already demonstrated for a variety of

Lipids bearing an alcohol function were ﬁrst esteriﬁed with

glutaric anhydride to provide the corresponding hemiglutaryl

acid, which was then activated with ethyl chloroformate as

described above or directly coupled using pyBOP as activating

agent (Figure 4).

Powder X-ray Diﬀraction Analysis. Crystallinity of the

synthesized prodrugs has been investigated by powder X-ray

diﬀraction, and d-spacing values are reported in Table S1. A

similar pattern was observed for the SQGem and RSQGem

prodrugs. At low angles, both conjugates showed two sharp

peaks (a main one and one on the right), corresponding to ﬁrst

46,47

amide-bearing prodrugs,

bine conjugate. Overexpression of these enzymes in several

including the squalene−gemcita-

malignancies compared to healthy tissues would favor

selective and controlled drug release at the tumor site.

Bioconjugate Chem. 2021, 32, 782−793

Article

Figure 5. Powder X-ray pattern of gemcitabine prodrugs at (a,c) low angles (1.3 < 2θ_C_u< 5°) and (b,d) high angles (3.5 < 2θ_C_u< 50°).

and second orders of the successive (00l) Bragg reﬂections of a

lamellar structure of d-spacing 44.5 and 47.3 Å for SQGem and

RSQGem, respectively (Figure 5a and Table S1 ). The shoulder

observed on the left (d-spacing 46.6 and 50 Å) revealed the

coexistence of two lamellar phases in each prodrug, as

previously reported. In addition, a minor peak, correspond-

ing to a d-spacing of about 40 Å, was found on the right of each

ﬁrst order peaks (Figure 5a). Overall, these results indicated

that these two prodrugs could organize in a ternary mixture. At

higher angles, both prodrugs revealed a broad peak centered at

It must be noted that at high angles, the whole library

showed a broadening of the peaks which reﬂected the mean

spacing of chains devoid of long-range order in the plane of the

bilayer.

Overall, a one-dimensional lamellar crystalline packing (L_c)

was observed for all the prodrugs. When compared to other

structural studies of gemcitabine lipid prodrugs, lower d-

spacing values were herein observed, likely due to the presence

of not fully extended and more disordered hydrocarbon

chains. At higher angles, this disorder correlated with a main

.8 Å (Figure 5b and Table S1).

Low-angle XRD patterns of the other prodrugs revealed

broad peak with a typical d-spacing = 4.6 Å, as already

described for lecithins and stacks of phospholipid−

broad peaks, with variable intensity (Figure 5c and Table S1).

Whereas SolgluGem (magenta line) and C28Gem (green line)

exhibited (00l) Bragg lines up to an order of 3 and 2,

respectively, for the other prodrugs, only a single broad peak

cholesterol bilayers. The right asymmetry of these peaks

can be modeled by a broad peak centered around 4.2 Å,

corresponding to hexagonally packed hydrocarbon chains in

50,54,56

the gel phase.

(

order 1), with d-spacing values comprised between 44.1 and

1.5 Å, was observed. All these prodrugs showed a one-

Nanoparticle Preparation and Characterization. With

the already described SQGem prodrug (Table 1, #01) capable

of spontaneously self-assembling as nanoparticles at a

dimensional lamellar crystalline packing, and the width of the

peaks can be correlated with the inability of these compounds

to achieve a well-ordered long-range periodicity. SolgluGem

−

concentration of at least 1 mg·mL in water without addition

of any surface active agent, whether the other synthesized

prodrugs would display the same properties has been

investigated herein. After the best organic solvent for each

prodrug was identiﬁed (i.e., ethanol (EtOH), acetone (Ac), or

tetrahydrofuran (THF)), the corresponding solution was

added dropwise into water under stirring. Among the 10

investigated conjugates, four assembled spontaneously into

stable nanoparticles at the desired concentration. On the

contrary, despite tuning some experimental conditions (i.e.,

solvent nature, organic/aqueous phase ratio), the other lipid

prodrugs, including LAGem, immediately precipitated upon

(

magenta line) and DiglygluGem (aquamarine line) presented

the highest lamellar d-spacing values (67 Å or more),

characteristic of an interdigitated lamellar gel phase L_β. At

higher angles (Figure 5d and Table S1 ), apart from the

C28Gem (green line), these prodrugs showed a very broad

peak with d-spacing comprised between 5 and 4.5 Å and a

shoulder at around 4.3 Å < d-spacing < 4.0 Å. The existence of

several other peaks indicated the presence of one or more

crystalline phases within the planes of the lamella, as also

49,51,52

previously reported.

Bioconjugate Chem. 2021, 32, 782−793

culture medium supplemented with 10% fetal bovine serum at 37 °C. Values represents mean ± standard deviation (n = 3).

Article

Figure 6. Evolution of NP mean diameter and polydispersity index (PdI) after incubation in (a) water at room temperature and (b) L-15 cell

Figure 7. Representative cryo-TEM images of (a) SQGem, (b) RSQGem, (c) SolgluGem, (d) CholgluGem, and (e) VitEgluGem nanoparticles.

Scale bar = 100 nm.

0−74

the addition step or aggregated in the aqueous phase during/or

rapidly after solvent removal.

Recently, however, the nanoprecipitation of a DMSO

the drug pharmacokinetics, thus altering its eﬃcacy.

this context, a successful attempt to develop a prodrug

formulation in aqueous solvent would be extremely beneﬁcial

to face these drawbacks.

solution of LAGem conjugates has been reported as a strategy

to formulate these conjugates into nanoparticles. Never-

theless, no details on the ﬁnal concentration of the nano-

assemblies have been provided. In addition, prior to in vitro

and in vivo administration, these nanoassemblies were modiﬁed

by the incorporation of PEGylated phospholipids, thus raising

concerns about the stability of the initial LAGem nano-

formulation.

Formulated NPs displayed a mean diameter between 80 and

190 nm, a narrow size distribution, and a negative surface

charge (Table 1). These NPs were stable in water over 72 h at

room temperature (Figure 6a and Figure S23 ), whereas a slight

size growth was immediately observed in complete cell culture

medium, likely due to the adsorption of the serum proteins at

the NP surface (Figure 6b). The most important augmentation

was recorded for the RSQGem NPs. Cryogenic transmission

electron microscopy (cryo-TEM) imaging of these NPs

revealed that the RSQGem conjugates assembled into a

dispersion of linear ﬁbers (Figure 7b), whereas CholgluGem

and VitEgluGem NPs (Figure 7d,e) formed elongated twisted

ﬁlaments. As previously reported, the conjugation to an

isoprenoid chain resulted, on the contrary, in the formation

Although nanoprecipitation was the method of choice for a

suitable comparison with the SQGem prodrug, we also

investigated other strategies such as the emulsion−evapo-

ration (with SolgluGem, EPAGem, and DHAGem) or the

thin ﬁlm hydration technique (with DiglygluGem and

OAGem). However, the eﬀort was in vain, and not any of

these strategies allowed the formation of stable nanoassemblies

satisfying our requirements (that is, pure water as aqueous

40,45

of spherical NPs (Figure 7a,c).

−

phase and concentration of at least 1 mg·mL ).

X-ray di ﬀ raction pattern of the various prodrugs (Figure 5

and Table S1 ) did not highlight a correlation with their

capacity (or not) to self-assemble into stable nanoparticles.

Thus, we investigated whether another physicochemical

parameter, namely, their HLB, could play a key role. The

These results clearly explained why previously described

fatty acid prodrugs of gemcitabine were administered only as

0−62

organic solution in DMSO

or mixtures with surface active

57,63

agents such as Pluronic, polyethylene glycol,

EL (i.e., polyoxyethylene castor oil), or Tween 80.

Cremophor

31,65,66

HLB values, calculated according to the Griﬃn method,

spite of their utilization also in clinically approved formulations

ranged from 6.00 for the SolgluGem to 9.10 for the LAGem

(Table 1) and a trend has emerged that, for values above 8.34,

the lipid conjugates were not able to spontaneously self-

67−69

(e.g., Emend, Taxotere, PM101, Taxol),

these vehicles are

associated with undesired toxicity and could negatively aﬀect

Bioconjugate Chem. 2021, 32, 782−793

even in the micromolar range (Figure 9 ). The slower

Article

Figure 8. Comparison of HLB values between prodrugs able (Yes) or not (No) to self-assemble in form of nanoparticles in pure water using the

nanoprecipitation technique (scatter plot, median; ns: nonsigniﬁcant, **p < 0.0021, ****p < 0.0001 Mann−Whitney’s test). (a) Lipid prodrugs

synthesized by coupling the Gem to the lipid moieties in Figure 1. (b) Pool of all the gemcitabine lipid prodrugs previously synthesized by us

(

including (a)). (c) Pool of all nucleoside lipid prodrugs previously synthesized by us (including (b)). Structures and HLB of each prodrug used in

this ﬁgure are reported in Table S2 .

assemble as nanoparticles (Figure 8a). Nevertheless, due to the

limited number of prodrugs included in this ﬁrst analysis, it was

hard to extrapolate a general behavior. Accordingly, to better

understand whether the HLB value could be an indicator of

the ability of a conjugate to self-assemble into NPs, we

introduced in this study several prodrugs already synthesized in

our group over the past 15 years. We ﬁrst focused on the

gemcitabine prodrugs only (Figure 8b), and then we also

included the whole library of available nucleoside analogue

derivatives, which successfully self-organized into nanoparticles

by nanoprecipitation in pure water, at the minimum

lipoprotein receptor activity. As also previously observed, the

SQGem NPs showed a higher cytotoxicity comparatively to

79,84

the free drug (IC of 1.3 μM vs 2.7 μM).

On the contrary,

the other NP formulations displayed only mild cytotoxicity

concentration of 1 mg·mL⁻(Figure 8c).

45,76−79

In both

cases, we observed a signiﬁcant diﬀerence between the HLB

values median. In the last group, which included the larger

number of prodrugs, these medians were of 7.45 and 9.07 for

the prodrugs which self-assembled or not, respectively (Figure

c).

Overall, these results conﬁrmed that an HLB below 8.34

that is, the lowest value in the group that does not form

Figure 9. Cell viability of MDA-MB-231 cells after incubation with

increasing concentrations of free Gem, SQGem, RSQGem,

SolgluGem, CholgluGem, and VitEgluGem nanoparticles. Values

represent mean ± standard deviation (n ≥ 3).

(

nanoparticles) could be a good indicator of the category to

which the prodrug belongs. In agreement with this hypothesis

is the observation that other gemcitabine prodrugs described in

literature (i.e., research groups other than ours) and reported

as unable to form stable NPs had an HLB above this critical

34,53

value (Table S3).

gemcitabine release from the SolgluGem NPs, herein selected

as an example of a nanoformulation with a low activity

comparatively to the SQGem NPs, is probably responsible for

the limited biological eﬃcacy (Figure S24 ). These results

showed that, in spite of the capacity of these prodrugs to

assemble as nanoparticles, the replacement of the squalene

chain with other lipid moieties seemed to be, at least in vitro,

therapeutically irrelevant.

The identiﬁcation of few exceptions (i.e., C28Gem (#11)

and the VitEgluGem (#05)) suggests that the HLB value

although predictive is, however, not suﬃcient alone to

determine whether a conjugate is able or not to form

nanoassemblies. Indeed, multiple other parameters such as π-

stacking and hydrogen bonding could aﬀect the self-

organization process, as previously observed with various

0−83

nucleolipids.

Yet, in vivo, these nanoparticles might show a diﬀerent

behavior. It is well-known that lipophilic drugs can bind to

lipoproteins, so it is possible to speculate that the HLB value of

the prodrugs would aﬀect their distribution proﬁle among

diﬀerent plasma components (lipoproteins, proteins) and

A close regard to the lipid chain of the prodrugs that

successfully assembled as nanoparticles revealed a shared

isoprene-like structural unit showing branched methyl groups

on the main carbon backbone. The latter are likely to hinder

the establishment of too strong hydrophobic interactions

between the prodrug molecules, which could spatially organize,

thus leading to the observed NPs. On the contrary, during the

nanoprecipitation a close packing of the other prodrugs

probably occurred with the formation of solid insoluble

aggregates.

consequently their therapeutic activity.

However, as previously observed with various molecular

86,87

species,

a prediction of the fate of gemcitabine prodrugs

based on hydrophobicity only could be rather reductive. In

addition to the HLB, other parameters such as binding

constants, relative abundance of plasma components, as well as

the colloidal stability of the nanocarriers into the blood

circulation, should be studied in depth in order to extrapolate a

broad structure−activity relationship.

In Vitro Studies. The cytotoxicity of the formulated NPs

was investigated on the MDA-MB-23 cells, a well-known

model of aggressive breast cancer characterized by a high

Bioconjugate Chem. 2021, 32, 782−793

Bioconjugate Chemistry

Article

CONCLUSIONS

(ChemAxon, 2021). The HLB was calculated with the HLB

Predictor plug-in using the Griﬃn empirical formula: HLB =

■

Using a small library of amphiphilic lipid prodrugs of

gemcitabine, we investigated how the nature of the lipid

moieties could aﬀect the capacity (or not) to self-assemble as

nanoparticles in pure water without the addition of any

surfactant. The HLB value was retained as a valuable predictive

parameter. Indeed, although with some exceptions, conjugates

having an HLB over 8.34 could not be formulated as stable

nanoparticles. The presence of lateral methyl on the hydro-

phobic chain could also be a key parameter in the ability of the

bioconjugates to self-assemble. However, the nanoparticulate

form was not the guaranty, at least in vitro, of a relevant

biological activity since the NPs other than SQGem displayed

only moderated cytotoxicity comparatively to the free drug and

to the SQGem NPs, which once again conﬁrmed their eﬃcacy.

Overall, these ﬁndings might help the design of optimal

conjugates which, thanks to their self-assembling ability, could

be formulated as nanoparticles without the need of using

potentially toxic vehicles.

0 (1 − S/A), where S and A correspond to the saponiﬁcation

and the acid numbers of the molecule, respectively.

DRX Analysis. X-ray diﬀraction (XRD) experiments were

carried out on a high-resolution θ−θ powder diﬀractometer

(

D8 Advance, Bruker AXS, Germany) equipped with the

LynxEye XE-T detector (1D mode), dynamic beam

optimization module (combining the use of variable slits,

automatic antiscatter screen position and controlled detector

opening) and a Cu radiation (K = 1.5406 Å and K = 1.5445

α1

α2

Å). The former drastically reduced the diﬀusion signal at very

low angles (2θ_C_u< 10°), and combined with a careful control

of the sample height, it allowed reproducible qualitative

information to be obtained on samples exhibiting d-spacing up

to about 68 Å. All prodrugs were dissolved into an optimal

organic solvent (that is, DCM (SQGem, SolgluGem

VitEgluGem, DiglygluGem, LAGem, EPAGem DHAGem);

EtOH (RSQGem and OAGem), or THF (CholgluGem and

C28Gem)) at a concentration of either 12.5 or 25 mg·mL⁻

depending on their solubility. Next, 2.5 mg was deposited onto

zero background silicon wafers (24 mm diameter, Siltronix,

France). Solvent was evaporated at room temperature prior to

analysis, resulting in a thin ﬁlm of the lipid prodrugs onto the

wafer surface. Samples were analyzed at room temperature

with a 10 rpm rotation, 2.5° Sollers slits, and a sample

illumination surface ﬁxed at 10 mm (variable slits mode). XRD

patterns were acquired at two diﬀerent theta ranges: (i) from

1.3 to 5° (2θ range) with a step size of 0.03° and 5 s per step

(n = 3 per prodrug), an antiscatter screen ﬁxed at 0.5 mm from

the surface of the ﬁlm, a limited detector opening (PSD = 1°)

and (ii) from 3.5 to 50° (2θ range) with an antiscatter screen

position automatically optimized by the software, a full

detector opening, with a step size of 0.03° and 4 s per step

(n = 1 per prodrug).

EXPERIMENTAL PROCEDURES

■

Materials. Regular analytical grade and deuterated solvents

were supplied by Carlo Erba (France) and Eurisotop (France),

respectively. Tetrahydrofuran was distilled from sodium/

benzophenone ketyl. Toluene, pyridine, dimethylformamide

(

DMF), and dichloromethane (DCM) were distilled from

calcium hydride, under a nitrogen atmosphere. All reactions

involving air- or water-sensitive compounds were routinely

conducted in glassware which was ﬂame-dried under a positive

pressure of nitrogen. Gemcitabine (Gem) hydrochloride,

gemcitabine base, solanesol (Sol), eicosapentaenoic acid

EPA), docosahexaenoic acid (DHA), and 4-dimethylamino-

pyridine (DMAP) were purchased from Carbosynth Ltd.

UK). Squalene (SQ), cholesterol (Chol), vitamin E (VitE),

(

oleic acid (OA), linoleic acid (LA), octacosanoic acid (C28),

glutaric anhydride, benzotriazole-1-yl-oxytrispyrrolidino-

phosphonium hexaﬂuorophosphate (pyBOP), ethyl chlorofor-

mate, N,N-diisopropylethylamine (diPEA), triethylamine, tert-

butyldimethylsilyl chloride (TBDMSCl), imidazole (Im), and

tetra-n-butylammonium ﬂuoride (TBAF) were purchased from

Sigma-Aldrich (France). Chemicals obtained from commercial

suppliers were used without further puriﬁcation.

Analytical Techniques. Infrared (IR) spectra were

obtained as solid or neat liquid on a Fourier transform Bruker

Vector 22 spectrometer. Only signiﬁcant absorptions are listed.

Optical rotations were measured on a PerkinElmer 241

NP Formulation and Characterization. Lipid prodrug

nanoparticles were prepared according to the nanoprecipita-

8,88

tion methodology (Table 1).

Brieﬂy, prodrugs were

−

dissolved at 2 mg·mL in a water-miscible organic solvent

(EtOH for SQGem, RSQGem, OAGem, LAGem, EPAGem,

DHAGem; acetone for CholgluGem, OAGem, LAGem,

EPAGem, DHAGem; THF for SolgluGem, VitEgluGem,

DiglygluGem, OAGem, LAGem, EPAGem, DHAGem,

C28Gem) (Table 1). Five hundred microliters of the resulting

solution was then added dropwise to a stirred solution of Milli-

Q water at solvent to water ratio of 1:2 v/v (SQGem,

RSQGem, SolgluGem, CholgluGem, DiglygluGem, OAGem,

LAGem, EPAGem, DHAGem, and C28Gem) or 1:4 v/v

(VitEgluGem). After removal of the organic solvent under

reduced pressure, an aqueous dispersion of NPs at a ﬁnal

polarimeter at 589 nm. The H and C NMR spectra were

recorded on Bruker Avance 300 (300 and 75 MHz for H and

C, respectively) or Bruker Avance 400 (400 and 100 MHz

−1

for H and C, respectively) spectrometers. Recognition of

methyl, methylene, methine, and quaternary carbon nuclei in

C NMR spectra rests on the J-modulated spin−echo

sequence. Mass spectra (MS) and high resolution mass spectra

HRMS) were recorded using electrospray ionization (ESI) on

concentration of 1 mg·mL was (or not) obtained. In order to

reach this value, VitEgluGem NPs were concentrated under

vacuum by removal of 1 mL of water.

When NPs were obtained, mean diameter and size

distribution were measured by dynamic light scattering with

a Nano ZS from Malvern (173° scattering angle) at 25 °C. The

measurements were performed after NP dilution at 0.1 mg·

(

an LTQ-Velos Pro mass spectrometer (ThermoScientiﬁc,

Germany). Analytical thin-layer chromatography was per-

formed on Merck silica gel 60F₂₅₄glass precoated plates (0.25

mm layer). Column chromatography was performed on Merck

silica gel 60 (230−400 mesh ASTM).

Hydrophilic Lipophilic Balance Calculations. The

hydrophilic−lipophilic balance values of the lipid prodrugs

were determined using MarvinSketch version 21.3 software

−

mL in Milli-Q water. The NP surface charge was investigated

by ζ-potential measurement at 25 °C after dilution at 0.05 mg·

−

mL in 1 mM NaCl solution applying the Smoluchowski

equation and using the same apparatus. Measurements were

carried out at least in triplicate. Colloidal stability has been

assessed by measuring NP mean diameter and size distribution

Bioconjugate Chem. 2021, 32, 782−793

Bioconjugate Chemistry

The Supporting Information is available free of charge at

Article

over a period of 72 h. NPs were diluted to 0.1 mg·mL⁻¹in

Milli-Q water or in complete cell culture medium (L-15

Leibovitz supplemented with 10% fetal bovine serum) and

incubated at 4 °C and room temperature or at 37 °C,

respectively.

nm using a microplate reader (LAB System Original Multiscan

MS). The percentage of viable cells in each well was calculated

as the absorbance ratio between treated and nontreated control

cells. All experiments were set up at least in triplicate to

determine means and standard deviation (SD).

Cryogenic Transmission Electron Microscopy. NP

morphology has been examined by cryo-TEM. Brieﬂy, 5 μL

of NP dispersion at a concentration of 1 mg·mL⁻for SQGem,

RSQGem, SolgluGem, and VitEgluGem NPs and of 0.5 mg·

mL for CholgluGem NPs were deposited onto a 200-mesh

holey carbon copper grid (TedPella Inc.) and ﬂash-frozen in

liquid ethane cooled at liquid nitrogen temperature. Cryo-

TEM images were acquired on a JEOL 2200FS energy-ﬁltered

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00051.

■

−

Detailed synthesis of gemcitabine lipid prodrugs; NMR

spectra; zeta-potential of nanoparticles over time;

gemcitabine release from SQGem and SolgluGem

NPs; d-spacing of gemcitabine lipid prodrugs; list of

the lipid prodrugs investigated in Figure 8; list of the

lipid prodrugs described in literature, which do not form

NPs by nanoprecipitation (PDF)

(

20 eV) ﬁeld emission gun electron microscope operating at

00 kV using a Gatan ssCCD 2048 × 2048 pixels.

Gemcitabine Release. The release of gemcitabine from

SQGem and SolgluGem NPs was investigated in the presence

of serum according to a previously described procedure with

89,90

slight modiﬁcations.

Practically, nanoparticles (200 μL)

AUTHOR INFORMATION

Corresponding Author

were incubated with 800 μL of human serum (Sigma-Aldrich,

■

France) supplemented with 570 mg·mL⁻tetrahydrouridine at

7 °C with gentle shaking. At predetermined time points (t =

, 1, 2, 4, 8, and 24 h), aliquots (100 μL) were withdrawn and

Simona Mura − Institut Galien Paris-Saclay, UMR 8612,

CNRS, Université Paris-Saclay, Faculté de Pharmacie, F-

92296 cedex Cha tenay-Malabry, France; orcid.org/0000-

0003-0310-8052 ; Email: simona.mura@universite-paris-

saclay.fr

spiked with 10 μL of the internal standard solution

theophylline, 10 μM in Milli-Q water) before addition of 1

(

mL of a mixture of acetonitrile/methanol (90:10, v/v) and

ultracentrifuged (20 min, 4 °C, 15000g). The supernatant was

then evaporated to dryness at 30 °C under a nitrogen ﬂow.

The released drug was quantiﬁed by reverse-phase HPLC. The

chromatographic system consisted of a Waters 1525 Binary LC

pump, a Waters 2707 Autosampler, a Waters 2998 program-

mable photodiode array detector (Waters, Milford, MA 01757,

USA) and a C18 Uptisphere column (3 μm, 150 × 4.6 mm;

Interchim). The HPLC column was maintained at 30 °C.

Detection was monitored at 270 nm. The HPLC mobile phase

consisted of a mixture of methanol/water with 0.05 M sodium

acetate (pH 5; solvent A: 5/95, v/v; solvent B 97/3, v/v). The

residues were dissolved in solvent A (100 μL). Elution was

performed at a ﬂow rate of 0.8 mL/min isocratically for 11 min

with solvent A followed by a linear gradient (1 min) to 75%

solvent A. After a 6 min hold, a 100% solvent B was achieved

by a linear gradient (1 min) and kept for 10 min. After a linear

gradient (1 min) to 100% solvent A, the system was held for 5

min for equilibration back to initial status.

Authors

Eleonore Coppens − Institut Galien Paris-Saclay, UMR

612, CNRS, Université Paris-Saclay, Faculté de Pharmacie,

F-92296 cedex Chatenay-Malabry, France

Didier Desmaële − Institut Galien Paris-Saclay, UMR 8612,

CNRS, Université Paris-Saclay, Faculté de Pharmacie, F-

2296 cedex Chatenay-Malabry, France

Julie Mougin − Institut Galien Paris-Saclay, UMR 8612,

CNRS, Université Paris-Saclay, Faculté de Pharmacie, F-

2296 cedex Chat

Sandrine Tusseau-Nenez − Laboratoire de Physique de la

Matiere Condensée (PMC), CNRS, Ecole Polytechnique,

enay-Malabry, France

Institut Polytechnique de Paris, 91120 Palaiseau, France

Patrick Couvreur − Institut Galien Paris-Saclay, UMR 8612,

CNRS, Université Paris-Saclay, Faculté de Pharmacie, F-

92296 cedex Chatenay-Malabry, France

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.bioconjchem.1c00051

Cell Culture. Human breast cancer cells MDA-MB-231

were obtained from the American Type Culture Collection and

cultured as recommended. Brieﬂy, cells were maintained in

Leibovitz’s L15 medium supplemented with 15% fetal bovine

Notes

The authors declare the following competing ﬁnancial

interest(s): Patrick Couvreur is co-founder of Squal Pharma

https://squalpharma.com/.

−

−1

serum, 2 mg·mL sodium bicarbonate, 50 U·mL penicillin,

−

0 μg·mL streptomycin, and 2 mM glutamine. Cells were

maintained in a humid atmosphere at 37 °C with 5% CO₂.

Cytotoxicity Evaluation. MDA-MB-231 cells were seeded

in 96-well plates (5000 cells per well in 100 μL of complete

culture medium) 24 h prior to exposure of a series of

concentration of NPs or free Gem for 72 h. Cell viability was

determined based on the mitochondrial dehydrogenase activity

using the 3-[4, 5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazo-

lium bromide (MTT) test. At the end of the incubation period,

ACKNOWLEDGMENTS

■

This work was supported by the French Ministry of Research,

the Fondation pour la Recherche Médicale (Application

FDT202001010776) and the Ligue contre le cancer

(Subvention Recherche HB2019-22 and NH2020-32). The

CNRS is also warmly acknowledged for ﬁnancial support. We

acknowledge Dr. Audrey Solgadi and Bastien Prost (UMS-

IPSIT-Plateforme SAMM, France) for mass spectrometry

analysis, the Electron Microscopy Facility of the Multimodal

Imaging Centre at Institut Curie, Orsay, for providing access to

the electron microscope, and Dr. Sylvain Trépout (Institut

Curie, France) for the cryo-TEM images. We would also like to

−

0 μL of a MTT solution (5 mg·mL in PBS) was added to

each well, and plates were incubated for 2 h at 37 °C.

Subsequently, the medium was gently removed, and the

formazan crystals were dissolved in dimethyl sulfoxide (200 μL

per well). Absorbance of the dye solution was measured at 570

Bioconjugate Chem. 2021, 32, 782−793

Bioconjugate Chemistry

Han, X., Du, Y., Li, L., et al. (2016) Self-Assembled Redox Dual-

Article

thank Miss Stéphanie Denis and Miss Clélia Mathieu (Institut

Galien Paris-Saclay, France) for their support in cell culture

experiments, and Miss Maëlle Mouzard for her help in the

chemical synthesis as part of her L2 internship.

(21) Sparreboom, A., Wolff, A. C., Verweij, J., Zabelina, Y., van

Zomeren, D. M., McIntire, G. L., Swindell, C. S., Donehower, R. C.,

and Baker, S. D. (2003) Disposition of docosahexaenoic acid-

paclitaxel, a novel taxane, in blood: in vitro and clinical

pharmacokinetic studies. Clin. Cancer Res. 9, 151−159.

(22) Luo, C., Sun, J., Liu, D., Sun, B., Miao, L., Musetti, S., Li, J.,

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