El
“P
an
Re Pr D C Prlaborato de
Peptide-ba
nd applica
Relatori rof. Fabio Be ott. Giovann ontrorelato rof. LorenzoDipar
Classe
el tirocinio
ased targe
ation to co
eltram ni Signore ore di BariU
NIVERSrtimento d
Corso
Curricu
e LM-54 (S
o di tesi:
eted stealth
ontrast-enh
SITÀ DEGLIdi Chimica
o di laurea m
C
HIMIA.A.
2013/
ulum: Chim
Scienze e T
h liposom
hanced ul
IS
TUDI DIP
e Chimica
magistrale
CA/2014
mico Organ
Tecnologie
mes: synthe
trasonogr
P
ISAIndustrial
in
nico
e Chimiche
esis, in vitr
raphy
”.
le
e)
ro evaluat
Ca Alessandrion
andidato ro RanalliThis thesis is dedicated to family.
To old families, new families and families yet to be born.
To families of relatives, families of lovers and families of friend.
Thanks and acknowledgements
This work would have not seen the light of day without the guidance, expertise and (especially) patience of doctor Giovanni Signore, my supervisor at NEST.
Thank you also to professor Fabio Beltram, my senior supervisor, for his guidance and his willingness to review my work on such a short notice.
A big thank you to my labmates for their support and help throughout these months. A special mention to Luigi, who helped me with the peptide syntheses and characterizations, to Melissa, who took care of the cell cultures and assisted me in flow cytometry analyses and to Francesco, who taught me how to use ImageJ.
Thank you also to doctor Bishuc and Geblin, for their help in reviewing the literature and doctor C., who has been at my side since the beginning of my career.
Summary
Efficiency of conventional antitumor therapies is often limited by unfavorable pharmacokinetics of conventional drugs, which usually show significant off-target activity, poor internalization in cells, and scarce retention in the host system. Nanomedicine aims at overcoming most of these limitations by allowing targeted, effective delivery of drugs and/or contrast agents, with the final goal of performing combined diagnostic and therapeutic activity with a single system, an approach referred to as theranostics; to this end, several nanostructures have been studied, but organic-based assemblies such as liposomes or lipoplexes are particularly attractive owing to their easy preparation, biocompatibility, and biodegradability.
Liposomes have been studied as drug carriers since the 1960s, owing to their ability to convey drugs to tumor tissues by passive accumulation, limiting side effects which are often encountered when dealing with free drugs. Notably, liposomal drug formulations are routinely used in a clinical setting. Efficiency of liposomal-based therapy can be significantly enhanced through insertion of poly-(ehtyleneglycol) residues, which inhibit protein adsorption and prevent opsonization, thus increasing residence time of the carrier in the bloodstream. An attractive improvement in liposome-based therapy is represented by the insertion of functional units that promote active targeting of a specific cell or tissue. Indeed, use of antibody-, ligand-, or peptide-derivatized liposomes represents a promising integration of already assessed delivery technologies with innovative targeting components.
In this work we developed a new class of liposomes tailored to in vivo targeted delivery and diagnostic. Our structures were assembled starting from commercially available lipids and novel lipopeptides, and their physic-chemical properties were evaluated in vitro and in living cells. We rationally designed and synthesized lipopeptides able to i) perform effective targeting of transferrin receptor, a membrane protein overexpressed by most tumor cells, and ii) hamper aspecific adsorption of serum proteins on the surface of the liposome, a key step in the recognition and clearance of nanostructures by the immune system.
Peptide-based liposomes were examined in detail, both in controlled conditions (cuvette) to assess their stability in various media, and in vitro on a model cell line of human pancreatic carcinoma (MIA PaCa-2) to investigate their biological activity; in both cases they compare favorably with plain, lipid-based liposomes already described in the literature.
Finally, the liposomes were conjugated to microbubbles, a contrast agent widely used in ultrasound based imaging, and their binding to MIA PaCa-2 cells was quantitated through flow cytometry.
We found that targeting properties of liposomes are fully retained upon conjugation to microbubbles, and thus they effectively recognize tumor cells. In perspective, our liposome-coated microbubbles can be regarded as a promising tool for in vivo theranostics.
Contents
Thanks and acknowledgements ... II Summary ... III List of tables ... IX List of figures ... X List of abbreviations and acronyms ... XII
1 Introduction ... 1
1.1 Liposomes ... 1
1.1.1 Liposome classification ... 2
1.1.2 Liposome preparation ... 2
1.1.3 Liposomes in drug delivery ... 4
1.1.4 Stealth liposomes ... 5
1.1.5 PEG alternatives ... 7
1.1.6 Targeted liposomes ... 8
1.2 Microbubbles and Contrast Ultrasound ... 10
1.2.1 Modern agents ... 10
1.2.2 Microbubble in drug delivery ... 11
1.3 Peptide Aptamers ... 12
1.3.1 Transferrin and its receptors ... 13
1.3.2 Peptide aptamers that target the transferrin receptor ... 15
2 Results and discussion ... 16
2.1 General remarks ... 16
2.2 Peptide and lipopeptide synthesis ... 18
2.2.1 Targeting peptides ... 18
2.2.2 Labeling of targeting peptide T7 ... 18
2.2.3 T7-Atto633 and MIA PaCa-2 cells ... 20
2.2.4 Targeting lipopeptide Pep-T7... 20
2.2.5 Stealth lipopeptides ... 22
2.3 Liposome synthesis ... 24
2.3.2 Stealth Liposomes ... 28
2.3.3 Stability in fetal bovine serum ... 31
2.3.4 Stealth and targeted liposomes ... 33
2.3.5 Stealth, targeted and linkable liposomes ... 36
2.4 Microbubble synthesis ... 39
2.4.1 μB-1 – Simple Microbubbles ... 39
2.4.2 Visualization of microbubbles with confocal microscopy ... 41
2.4.3 μB-2 – maleimide-linkable microbubbles ... 42
2.4.4 μB-3 and μB-4 – Microbubbles with stealth and targeted liposomes ... 43
2.5 Confocal microscopy experiments ... 44
2.5.1 Simple liposomes and MIA PaCa-2 cells ... 44
2.5.2 Stealth liposomes and MIA PaCa-2 cells ... 44
2.5.3 Stealth/targeted liposomes and MIA PaCa-2 cells ... 45
2.6 Flow cytometry experiments ... 47
2.6.1 Microbubble experiments ... 47
2.6.2 Liposome experiments... 50
3 Conclusions and future perspectives ... 53
4 Material and methods ... 55
4.1 General ... 55
4.1.1 Chemicals and consumables ... 55
4.1.2 Buffers and hydration solutions ... 55
4.1.3 Peptide synthesis ... 55
4.1.4 HPLC and HPLC-MS analyses ... 56
4.1.5 Sonication ... 56
4.1.6 DLS and Zeta-potential measurements ... 56
4.1.7 Cell culture ... 56
4.1.8 Confocal microscopy imaging ... 57
4.1.9 Flow-cytometry ... 57
4.2 Peptide Synthesis ... 58
4.2.2 T7: Targeting peptide ... 60
4.2.3 T7-GC: Targeting peptide, linkable ... 60
4.2.4 Pep-T7: Targeting lipopeptide ... 61
4.2.5 Pep-Stealth: Stealth lipopeptide ... 62
4.2.6 Pep-StealthCys: Stealth lipopeptide, linkable ... 62
4.2.7 T7-Atto633 ... 63
4.3 Liposome synthesis ... 65
4.3.1 Liposome loading ... 65
4.3.2 Measurement of non-encapsulated doxorubicin – general procedure ... 65
4.3.3 Size measurement by DLS ... 66
4.3.4 Characterization: measurement of the Zeta potential ... 66
4.3.5 Lipo-1: Simple liposome ... 66
4.3.6 Lipo-2: Simple liposome with DPPG ... 66
4.3.7 Lipo-3: Stealth liposomes ... 67
4.3.8 Lipo-4: Stealth liposome (1:7), untargeted ... 67
4.3.9 Lipo-5: Stealth liposome (1:7), targeted ... 67
4.3.10 Lipo-6: Stealth liposome (1:7), targeted, maleimide-linkable ... 68
4.3.11 Lipo-7: Stealth liposome (1:7), targeted, stealth-linkable ... 68
4.4 Liposome stability tests ... 68
4.5 Microbubble synthesis ... 69
4.5.1 General ... 69
4.5.2 μB-1 – Simple microbubbles ... 69
4.5.3 μB-2 – maleimide-linkable microbubbles ... 69
4.5.4 Microbubble sizing ... 69
4.5.5 Microbubble coupling – general procedure ... 70
4.6 T7-Atto633 internalization experiment ... 71
4.7 Liposome internalization experiments ... 71
4.8 Microbubbles adhesion experiments ... 71
List of tables
Table 1 - Survey of targeted liposomes in advanced phases of trial. ... 9
Table 2 - Selected ultrasound contrast media available on the market ... 10
Table 3 - Size distribution of stealth liposomes Lipo-3. ... 29
Table 4 – Changes in size of Lipo-3, after 120 h of storage, as measured by DLS. ... 31
Table 5 - Residues and their respective synthetic equivalents ... 58
Table 6 - Mass spectrometry data for T7 ... 60
Table 7 - Mass spectrometry data for T7-GC ... 61
Table 8 - Mass spectrometry data for Pep-T7 ... 62
Table 9 - Mass spectrometry data for Pep-Stealth ... 62
Table 10 - Mass spectrometry data for Pep-StealthCys ... 63
Table 11 - Mass spectrometry data for T7-Atto633 ... 64
Table 12 - Shell composition of stealth liposomes Lipo-3 ... 67
List of figures
Figure 1 – Illustration of the process through which liposomes assemble to yield
phospholipids. ... 1
Figure 2 - Size and morphologies of different liposomes... 2
Figure 3 - Selected methods of preparation of liposomes. ... 3
Figure 4 - Scheme of the process that leads to MLVs... 3
Figure 5 – Picture of the Mini-Extruder manufactured by Avanti Polar Lipids.. ... 4
Figure 6 - Regimens of grafted PEG over the surface of liposomes, as proposed by de Gennes.31 ... 7
Figure 7 – An overall diagram of Tf trafficking ... 13
Figure 8 - Examples of strategies for targeting therapeutic agents via TfR to malignant cells. .. 14
Figure 10 - HPLC chromatogram for the synthesis of T7-GC ... 19
Figure 11 - Internalization of T7-Atto633 in MIA PaCa-2 cells. ... 20
Figure 12 - Design of the lipopeptide Pep-T7. ... 21
Figure 13 - HPLC chromatogram of crude Pep-T7 ... 21
Figure 14 - Structure of Pep-Stealth ... 23
Figure 15 – HPLC chromatogram of crude Pep-Stealth ... 23
Figure 16 - Scheme of the liposome preparation procedure. ... 24
Figure 17 - Size distribution for simple liposomes "Lipo-1", as measured by DLS. ... 26
Figure 18 - Size distribution of liposomes "Lipo-1" after 1 week of storage, as measured by DLS. ... 27
Figure 19 - Size distribution for simple liposomes with DPPG "Lipo-2”, as measured by DLS 27 Figure 20 - Size distribution of stealth liposomes Lipo-3, measured after their synthesis by DLS. ... 29
Figure 21 – Stealth lipids in a non-skewed configuration (left panel) and in a skewed one (right panel) ... 30
Figure 22 - Size distribution of Lipo-3, after 120 h of storage as measured by DLS. ... 30
Figure 23 - Size distribution of Lipo-3c (stealth liposomes with 25% of Pep-Stealth) after incubation with solutions of FBS at different concentrations. ... 32
Figure 24 – Size distribution of simple liposomes Lipo-2, after 20 minutes of incubation in FBS as measured by DLS. ... 32
Figure 25 – Mean diameter of Lipo-5, as measured by DLS at different temeperatures. ... 34
Figure 26 - Standard deviation in the diameter of Lipo-5, as measured by DLS at different temperatures. ... 34
Figure 27 - Mean diameter of Lipo-5 and Lipo-2 as a function of time, during incubation with undiluted FBS, as measured by DLS... 35
Figure 28- Standard deviation in diameter of Lipo-5 as afunction of time, during incubation with undiluted FBS, as measured by DLS. ... 36
Figure 29 - HPLC chromatogram of crude Pep-StealthCys ... 38
Figure 30 - Size distribution for unpurified simple microbubbles μB-1 ... 40
Figure 31 - Mechanism of the purification of microbubbles... 40
Figure 32 - Size distribution of μB-1 after purification, as measured by DLS. ... 41
Figure 33 - Microbubbles, directly visualized on a microscope slide. ... 42
Figure 34 – Confocal microscopy images of MIA PaCa-2 cells incubated for 30 minutes at 37 °C with Lipo-2. ... 44
Figure 35 – Confocal microscopy images of MIA PaCa-2 cells incubated for 30 minutes at 37 °C with Lipo-4. ... 45
Figure 36 - Confocal microscopy images of MIA PaCa-2 cells incubated for 30 minutes at 37 °C with Lipo-5.. ... 45
Figure 37 – Flow cytometry scattering plots of MIA-PaCa-2 cells. ... 47
Figure 38 - Scheme of the optical cell of a flow-cytometer. ... 48
Figure 39 - Plot of the fluorescence values for MIA PaCa-2 cells, both treated (red line) and untrated (black line) with μB-3. ... 49
Figure 40 - Plot of the fluorescence values for MIA PaCa-2 cells, both treated (blue line) and untrated (black line) with μB-4. ... 50
Figure 41 - Plot of the fluorescence values for MIA PaCa-2 cells, both treated (red line) and untrated (black line) with Lipo-4. ... 51
Figure 42 – Plot of the fluorescence values for MIA PaCa-2 cells, treated with untargeted stealth liposomes Lipo-4 (left, blue line) and with targeted stealth liposomes Lipo-5 (right, green line). ... 51
List of abbreviations and acronyms
CEUS: contrast-enhanced ultrasonography
CMC: critical micellar concentration
DAD: diode array detector
DIPEA: N,N-Diisopropylethylamine
DLS: Dynamic Light Scattering
DMF: Dimethylformamide
DOPE-Rhod: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)
DOX: Doxorubicin
DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine
EDT: 1,2-ethanedithiol
EG: ethylene glycol
EPR: enhanced permeation and retention
FBS: fetal bovine serum
FSC: forward scattering
GUV: giant unilamellar vesicle
HBTU: N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HPLC: high performance liquid chromatography
hTfR: human transferrin receptor
LUV: large unilamellar vesicle
MLV: multilamellar vesicle
NIR: near infrared
NMP: 1-methyl-2-pyrrolidinone
PDP: 2-pyrydildithiopropionamide group
PEG: polyethylene glycol
RES: rethiculo-endothelial system
SPPS: solid phase protein synthesis
SSC: side scattering
SUV: small unilamellar vesicle
TCEP: tris(2-carboxyethyl)phosphine
Tf: transferrin
TfR: transferrin receptor
1
1.
Li pa ha re Th hy aq ha ar ex Th is Th lea In ph ph caIntrod
.1 Lipos
iposomes w articles”, com ave a proper eview on the he bilayer of ydrophilic “h queous med ave critical m re four to fiv xtremely low he fundamen shown below Figure 1 – phospholipid he individua aflets (b), wh n a laborato hosphatidilch hosphatidyle ationic lipidsduction
somes
were first de mprised of a r name until topic.2 f liposomes head” and a dia, self-assem micelle conce ve orders of w. ntal process w (Fig. 1) Illustration of t ds; (b): phospho al phosphol hich then ev ory setting, holines, alo ethanolamin s such as stea escribed by bilayer mem 1968, when is most com a hydrophob mble into b entrations (C f magnitude through whthe process thro olipid bilayer; (
lipid molecu volve into lipo
the most c ong with th e and phos arylamines ca Bangham a mbrane whic n Sessa and W mmonly comp bic tail. The bilayer leafle CMCs) of 10
times small
hich liposom
ough which lipo (c): liposome; (
Weissig
ules (a) init osomes. commonly u he negative sphatidiylser an be used. and cowork ch surrounds Weissmann prised of ph se lipids, ow ets; however 0-2-10-4 M, th ler, meaning es form, star osomes assemb (d): detail of the g.3 tially aggreg used phosp phosphatid rine. When kers in 1961 s an aqueous coined the w ospholipids, wing to their r, whereas o he CMC of b g that their s rting from th le to yield phos e bilayer structu gate to form holipids are dic acids, p cationic lip 1 as “spheri s core.1 They word “liposo , molecules b r scarce solu ordinary amp bilayer-form solubility in he free phosp spholipids. (a): ure. Reproduce m bilayer m e the charg phosphatidy osomes are ical lipid y did not ome” in a bearing a ubility in phiphiles ing lipids n water is pholipids free ed from membrane ge-neutral ylglycerol, needed,
Th of ela in th 1. Fr th U th Th wi m th 1. Si fo m th he nature of f the bilayer asticity, its ncreases tran hese properti .1.1 Lipos rom a morph heir number Unilamellar li heir well-cha hey are som ith large and more studied heir behavior .1.2 Lipos ince liposom ormulations h methods have
his topic desc
f the fatty ac r: the overal permeability nsition tempe ies, most not
some classi hological po of lamellae ( Figure 2 - Size a iposomes be aracterized m mewhat arbitr d small vesic and used. M r is often unp some prepa mes were re has stimulat e been develo
cribe the resp
cid residue o ll phase beh y. For exam erature by 1 tably the mem
ification int of view, l (Fig. 2).4 and morpholog ear a special membrane b rarily divided cles (under Multivescicu predictable.4 aration ecognized a ed research oped and so pective proto of the lipids i havior (for in mple, elonga 10-15 °C. Th mbrane crys liposomes ar gies of different interest in r behavior, eas d into small 200 nm and ular and mul
as drug carr on preparat me of them ocols in deta is responsibl nstance, its ation of the he addition stallinity. re most frequ liposomes. Rep research and se of prepar l, large and g d 50 nm in d tilamellar lip riers in the tion procedu are outlined ail.5–7
le for the fun transition te e fatty acid of cholester uently classi produced from W d biomedical ration and th giant vesicles diameter res posomes stil first 1980s, ures. In the p d in Fig. 3. R ndamental p emperature chain by tw rol further m ified by their Weissig.3 l application herapeutic p s based on t spectively) b ll find limite s, the need past 30 year Reviews and b properties - Tc), its wo units modulates r size and ns, due to potential. their size, being the ed use, as for new s, several books on
Se aq In ch fre Th of ve Th (L ar te F everal fabric queous solut n these proce hloroform, c eeze-dried. he lipid film f choice abov esicles throug Figure 4 - Sche he MLVs pr LUVs or SUV re most freq echnique, wh Figure 3 - Selec ation metho ion, which m edures, the l chloroform/m or powder o ve the highes gh an “exfoli
eme of the proce action of water roduced this Vs) and seve quently synth hich is suitab ted methods of
ods start from may include t ipid mixture methanol, o obtained this st Tc of the li iation” mech
ess that leads to r (b), yielding m
s way must b eral method hesized by l le for lab use
f preparation of m the hydra the substanc e is dissolved or cyclohexa s way is hydr ipids in the m hanism, best o MLVs. A thin m multilamellar ve be then dow ds are availab low-pressure e but can also
f liposomes. Rep ation of a dr ce to be enca d into a suita ane) and is rated and m mixture. Thi described in multilayer of lip esicles (c). Adap wnsized to la ble to obtain e extrusion, o be implem produced from W ry lipid film psulated, to able, low-boi dried by a r echanically a is process pr n Fig. 4.
pids (a) is exfoli pted from Greg
arge or small either kind a robust an ented at the Weissig.3 with an exc produce ML iling solvent rotary evapo agitated with roduces mult iated by the me goriadis.7 ll unilamellar of liposome nd widely a industrial le ess of an LVs. t (usually orator or h a buffer tilamellar echanical r vesicles es.8,9 LUV pplicable evel.
In T po te us Th fo th m 1. Li bi dr ph U m fo Li bi en di
n the lab, the
Figure 5 – Pict enclose the The device i olycarbonate echniques, ar ser to produc he extrusion orcing the dis he second syr mixture, to avo .1.3 Lipos iposomes h iodegradable rugs) make hospholipids Unilamellar v main applicat ormulations o iposomal fo iodistributio ncapsulated iscontinuous extrusion pr
ture of the Mini sizing membra is made of e membran re available in ce a wide var n process is c spersion thro ringe. The p oid ruptures somes in dr have been e shell and th liposomes s are biologi vesicles rangi tion is as a of chemothe ormulations n throughou molecules f s endothelium rocess is carr -Extruder manu ne are clearly vi Repdoduce two syringe ne. These n different p riety of liposo carried out b ough the me process has t in the liposo rug deliver extensively heir aqueous an excellen cally inert an ing from 50 a vector for erapic drugs s of drugs ai ut the body a from degrad m, such as th
ried out usin
ufactured by Av isibile. The blac ed from Avanti P
es, separated membranes, pore sizes (ra omal dispers by drawing t embrane seve to be carried ome shells. ry studied as s internal env nt delivery nd weakly im nm to 250 r hydrophil such as doxo im to overc and targeting dation and c he liver, splee ng handheld d
vanti Polar Lipi ck base is made Polar Lipids we d by a set o , usually m anging from sions. the MLV dis eral times, co d out above t drug carri vironment (b system. Li mmunogeni nm are the lic drugs an orubicin (DO come two k g to specific r can passively en or bone m devices, like
ds. The two syr of steel and can bsite. of bolts and manufacture 30 nm to 50 spersion in t ollecting the the highest T iers. Their both capable posomes co c and have l most comm nd payloads OX) are wide
key problem receptors. Li y target tissu marrow.
the one in F
ringes and the b n act as a therm
d nuts that ed by track
00 nm) and
the first syrin e sample at th Tcof the lipi biocompat e of dissolvin omposed of low intrinsic monly used a s. Indeed, li ely available ms in drug iposomes pr ues or organ Fig. 5. bolts that mostat. encase a k-etching allow the nge, then he end in ids in the ible and ng several f natural c toxicity. and their iposomal . therapy: rotect the ns with a
On intravenous administration, liposomes made of simple phospholipids are rapidly recognized by the rethiculo-endothelial system (RES), particularly by the mononuclear phagocytes (MPS).10 These immune cells aim to clear liposomes from the bloodstream and may render liposomal therapy inefficient (unless cells from the RES are the actual target).11
The process through which the immune system recognizes liposomes usually starts with opsonization, i.e. the binding of selected serum proteins (opsonins) onto the surface of the liposomes. The MPS does not recognize the liposomes itself, but rather the opsonins on their surfaces and as of today a number of opsonizing proteins have been identified.12–15 Complement components12,16,17 are another important system able to recognize liposomes. This system acts by initiating membrane lysis (generating pores and the release of the liposome payload) and enhancing uptake by the RES cells.
The physical-chemical properties of liposomes, such as net surface charge, hydrophobicity, size and crystallinity of the membrane influence their stability and the type of proteins that bind to them. Therefore, the first attempts at reducing the MPS uptake focused on modulating the size and charge of the liposomes: smaller vesicles (SUVs) have significantly lower clearance than larger ones17 while no clear relation between charge and clearance has been established.18–21
1.1.4 Stealth liposomes
Several different strategies have been further developed to “hide” liposomes from the RES, enhancing residence time in the bloodstream.
The first approach studied was the preparation of liposomes mimicking the erythrocyte membrane, modifying the surface with gangliosides and sialic acid derivatives.22 Subsequent improvements aimed to increase the hydrophilicity of the liposomal surface by using hydrophilic polymers, which also allowed for a “steric stabilization” effect.
The mechanism whereby steric stabilization of liposomes increases their longevity in circulation has been extensively discussed.22 The basic concept is that a hydrophilic polymer or a glycolipid, such as PEG (polyethylene glycol) or GM1 (monosialotetrahexosylganglioside), possessing a flexible chain that occupies the space immediately adjacent to the liposome surface (“periliposomal layer”), tends to exclude other macromolecules from this space. Consequently, access and binding of blood plasma opsonins to the liposome surface are hindered and thus interactions of MPS macrophages with such liposomes are inhibited.
By reducing MPS uptake, long-circulating liposomes can passively accumulate inside other tissues or organs. This phenomenon, called passive targeting, is especially evident in solid tumors undergoing angiogenesis: the presence of a discontinuous endothelial lining in the tumor vasculature during angiogenesis facilitates extravasation of liposomal formulations into the interstitial space, where they accumulate due to the lack of efficient lymphatic drainage of
the tumor and function as a sustained drug-release system. This causes the preferential accumulation of liposomes in the tumor area (a process known as enhanced permeation and retention effect or EPR).23
Among the different polymers investigated in the attempt to improve the blood circulation time of liposomes, poly-(ethylene glycol) (PEG) has been widely used as polymeric steric stabilizer. It can be incorporated on the liposomal surface in different ways, but the most widely used method at present is to anchor the polymer in the liposomal membrane via a cross-linked lipid (i.e., PEG-DSPC).24
Moreover, compared to GM1, molecular weight and structure of PEG molecules can be freely modulated for specific purposes and it is easier and cheaper to conjugate the polymer with the lipid. Needham and coworkers also demonstrated that the presence of PEG on the liposome surface provides a strong interbilayer repulsion that can overcome the attractive Van der Waals forces, thus stabilizing liposome preparations by avoiding aggregation.25
Regarding MPS uptake, Blume and coworkers found that the increased blood circulation time is due to a reduced interaction with plasma proteins and cell-surface proteins,26,27 although other studies found no direct evidence of this reduced interaction with plasma components.28
A number of reports suggests that PEG does not completely avoid cumulative uptake by cells of the MPS and an interesting review updates progress in this area: Moghimi and coworkers critically examined the supposed mechanisms that contribute to prolonged circulation times of sterically protected liposomes.29 They point out that PEGylated liposomes are not completely biologically inert and that there is some evidence the polymer can still induce activation of complement systems: for instance PEGylated liposomal doxorubicin (DOXIL® in the US, Caelyx® in Europe), is a strong activator of the human complement system, with activation taking place within minutes.
The behavior of PEGylated liposomes depends on the physical-chemical properties of the specific PEG linked to the surface. Figure 6 represents the regimens proposed by deGennes, when polymers are attached to the liposome surface, depending on the length and graft density of the polymer.30
Th co In cir an pr no ex lo bi m 1. D alt PE m sy th Su m Figur he molecula overage and t n liposomes rculation life nd the lengt roduced the ot yet been xtend circula ngevity of d inding/uptak mechanism(s) .1.5 PEG espite the w ternative po EGylated lip modified drug ystem is cap herapy ineffic uitable polym main chain an re 6 - Regimens ar mass of the the distance composed o etime of the th or molec greatest imp established b ation lifetim drug carriers ke, there is ) by which su alternative well-develop olymers is on posomes and gs and drug able of prod cient, a phen
mers for lipo nd high bio of grafted PEG e polymer, as between gra of phospholi e vehicles wa cular weight provements between che e. Although by reducing s sufficient urface grafte es ped chemistr ngoing; this d by the hop carriers. Mo ducing Anti nomenon refe osomal coat compatibilit
G over the surfac
s well as the aft sites. ipids and ch as found to d t of the pol in blood res emical and p the accepte g or preventi conflicting ed PEGs imp ry of PEG s might be e pe of achievin oreover, rece i-PEG antibo ferred to as “a ting should ty. Synthetic ce of liposomes graft density holesterol, th depend on b lymer.31 In sidence time physical pro ed opinion i ing protein b data to w prove liposom coupled to explained bo ng even bett ent studies s odies,32 thus accelerated b be soluble, c polymers, , as proposed by y, determine he ability of both the am most cases, e. However, perties of PE s that PEG binding and/ warrant a r me propertie pharmaceut oth by the p ter control o showed that s rendering body clearan hydrophilic, such as poly y de Gennes.30 the degree o PEG to inc mount of graf , longer-cha a conclusive EG and its increases ci /or by inhib reassessment es.24 ticals, the se patent limita over the prop t the human long-term li nce”. , have highly ly(vinyl pyrr of surface rease the fted PEG ain PEGs e link has ability to rculation biting cell t of the earch for ations on perties of immune iposomal y flexible rolidone)
(PVP) and poly(acryl amide) (PAA), are the most prominent examples of other potentially protective polymers.33–35 Liposomes containing DSPE, covalently linked to poly(2-methyl-2-oxazoline) or to poly(2-ethyl-2-poly(2-methyl-2-oxazoline) also exhibit extended blood circulation time and decreased uptake by the liver and spleen.36 Similar observations were reported for phosphatidyl polyglycerols.37 More recent papers describe long circulating liposomes prepared using poly[N-(2-hydroxypropyl) methacrylamide]38amphiphilic poly-N-vinylpyrrolidones, 38 L-amino-acid-based biodegradable polymer–lipid conjugates,39 and polyvinyl alcohol.40 All groups of polymer-coated liposomes reported had an extended blood circulation time, while liver capture was diminished. These results are comparable with those for PEG liposomes; the efficacy of the steric effect quite naturally depends on the quantity of incorporated polymer.
1.1.6 Targeted liposomes
The use of targeted liposomes has been suggested to increase liposomal drug accumulation in the desired tissues, producing higher and more selective therapeutic activity. This strategy involves the coupling of targeting moieties capable of recognizing target cells, binding to them and inducing the internalization of liposomes or encapsulated drugs. Targeting moieties include monoclonal antibodies (MAb) or fragments, peptides, growth factors, glycoproteins, carbohydrates, or receptor ligands.41–43 Targeted liposomes offer various advantages over individual drugs targeted by means of polymers or antibodies. One of the most compelling advantages is the dramatic increase in drug amount that can be delivered to the target, avoiding cells and tissues, which are not of interest. Targeted liposomes also provide a “bystander killing” effect, because the drug molecules can diffuse into adjoining tumor cells.
A survey of therapeutic targets and their respective liposomal compositions that are in advanced phase of trial are reported in Table 1.
Targeting moiety Encapsulated drug Disease
Anti-HER2 (trastuzumab)44 Doxorubicin Breast, ovarian cancer
Anti-EGF45 Doxorubicin, vinorelbine Solid tumors
Anti CD1946 Vincristine Lymphoma
Anti CD22 anti-B47 Doxorubicin Cell lymphoma
Anti CD1948 Imatinib All
Anti-beta1 integrin49 Doxorubicin Several cancers
GAH MAb51 Doxorubicin Gastric, colon and breast cancer
Anti-EGF receptor52 RNA Brain cancer
Table 1 - Survey of targeted liposomes in advanced phases of trial.
Currently, the only immunoliposome formulation undergoing clinical trials is the PEGylated liposomal DOX, targeted with the F(ab')2 fragment of the human MAb GAH, which is able to recognize gastric, colon and breast cancer cells.53
1.2 Microbubbles and Contrast Ultrasound
Microbubbles are, per simple definition, gas-in-liquid emulsions with diameters ranging from 0.5 μm to 100 μm. Dilute air microbubbles occur naturally: they have been found in fresh54 and sea waters,55 as well as in blood and other bodily fluids by many investigators over the course of the last century.
The use of microbubbles as ultrasound contrast agents originated in the late ‘60s from a chance discovery by Dr. Claude Joyner, a cardiologist. While he was performing an M-mode echocardiogram, he noticed that the signal in the aortic root region increased for a brief amount of time just after the injection of saline solution, made via a cardiac catheter.56– 58Subsequent research revealed that the “agents” responsible for the increased signal-to-noise ratio were the free air bubbles, formed either at the tip of the catheter, or already suspended in the saline solution. This discovery led to a series of investigations over the next several decades and to the development of what is now simply referred to as CEUS (Contrast-Enhanced Ultrasound Sonography), ultimately leading to the commercial exploitation of microbubbles as ultrasound contrast agents.
1.2.1 Modern agents
Over the past 40 years, tremendous improvements have been done to bring microbubble formulations to the clinical use. The first group that managed to meet this challenge was Feinstein’s, in 1984,59 who produced stable microbubbles by sonicating a solution of human serum albumin and showed through direct microscopic visualization that they could pass unhindered through the capillary vasculature, after a peripheral injection. This agent was subsequently developed and marketed as Albunex® (Molecular Biosystems Inc., USA), the first FDA-approved agent for this use. Nowadays, there are several contrast agents available on the market; Table 2 shows a list of a few selected ones.
Name (producer) Core gas Shell composition
SonoVue (Bracco) SF6 Phospholipids (DPPC, phosphatidic acid)
Optison (GE Healthcare) C3F8 Albumin
Levovist (Schering) Air Lipids/Galactose
Definity (Lantheus MI) C3F8 Phospholipids
Table 2 - Selected ultrasound contrast media available on the market
Most of the microbubbles available on the market today contain fluorinated substances as the core gases. These media (known as “second generation”), overcome the issues generated by the excessive diffusivity of the air inside the microbubbles, which then readily dissolved in blood, resulting in a very short half-life (~ 1 min) and a poor contrast for most diagnostic applications.
1.2.2 Microbubble in drug delivery
It was shown that plasma membranes are temporarily permeabilized through the formation of transient pores in the cellular membrane when microbubbles collapse in the proximity of cells, a phenomenon called sonoporation.60 The use of microbubbles in combination with ultrasound may even induce openings in the blood-brain barrier which could be of interest to tackle drug delivery into the brain which currently remains a huge challenge.61,62
Effective ultrasound targeted drug delivery relies on the design of microbubbles which can be loaded with drug molecules. Basically, microbubbles can be loaded in three ways: (a) the drug can be incorporated in the microbubble shell,63 (b) (lipophilic) drugs can be incorporated in an inner oil phase present in the microbubbles64 or (c) “colloidally drug loaded microbubbles” can be obtained through the attachment of drug containing nanoparticles, like liposomes, on the microbubbles' surface as reviewed by Bohmer et al.65 and Lentacker et al.66
Loading the surface of microbubbles with drug containing liposomes is a promising concept for ultrasound guided drug delivery as: (a) more drugs can be loaded on microbubbles compared with other loading strategies, (b) a plethora of knowledge is available on liposomes for drug delivery which can be perfectly used to develop the colloidally loaded microbubble concept and (c) importantly, certain liposomes are safe and even already used in clinical practice.
Recently, the group of Geers and Lentacker proposed a series of (DOX)-liposome loaded microbubbles67 and lipoplex-loaded microbubbles (containing pDNA or siRNA),68 showing that in combination with ultrasound, such microbubbles strongly improved both doxorubicin (DOX) cytotoxicity and genetic material delivery to cells in vitro.
1.3 Peptide Aptamers
Strictly following the definition coined by Colas et al. in 1996,69 peptide aptamers are combinatorial protein molecules in which a variable peptide sequence with affinity for a given target protein is displayed on an inert, constant scaffold protein.69–74 They are extremely simple molecules, selected from combinatorial libraries on the basis of their affinity to the target protein or small molecule and expressed in bacterial cells, such as E. coli. Both termini of the variable sequence are fused to the inert scaffold, thus peptide aptamers are doubly constrained. This double constraint distinguishes peptide aptamers from other artificial combinatorial protein molecules, which often consist of random peptidic sequences fused terminally to a carrier protein or another macromolecule. Actually, the term does not comprise other types of double-constrained combinatorial proteins that are more complex than peptide aptamers because target-binding surfaces consist of noncontiguous peptidic sequences disseminated over several secondary structural elements or across several variable loops. 70,71 However, these double-constrained combinatorial proteins have similar characteristics and applications as peptide aptamers. In particular, all of them show molecular recognition properties, in a manner similar to antibodies, but with improved characteristics, such as small size, high stability, high solubility, high yield bacterial expression, possibility of chemical synthesis, rapid folding properties and in some cases, such as in the affibody molecules (affinity molecules based on the protein A scaffold), absence of disulfide bonds and of free cysteine residues. As reported by Löfblom and coworkers,75 the high stability in the absence of disulfide bonds is an important advantage, which facilitates high yields in bacterial expression and enables intracellular applications. Moreover, the absence of intramolecular cysteine residues gives the possibility of introducing a unique C-terminal cysteine residue for labeling or other chemical modifications. The final shape of these artificial constrained combinatorial proteins will be determined both by the amino acid composition and sequence of the peptide as well as by the primary sequence and tertiary structure of the scaffold protein.74 Importantly, binding affinity of these artificial proteins is greatly increased by the constraint applied by the scaffold and this is the main advantage associated with the use of conformationally constrained peptides versus unstructured linear peptides. Nevertheless, many peptide aptamers are still able to perform effective targeting even when not inserted in the scaffold protein. This greatly simplifies the synthetic approach necessary to insert a peptidic targeting sequence on a nanostructure.
Medical therapy and in vivo diagnostics are important fields of application for peptide aptamers.75–83 Several artificial combinatorial proteins are in preclinical studies and a few of them are in clinical trials.76 Theoretically, all the different artificial combinatorial proteins, provided that are able to bind a particular target molecule with sufficient affinity and selectivity, are applicable as bioreceptors in bioassays. To date, only a few studies have focused on the actual use in this regard.84 In comparison to what happens in the nucleic acid aptamer
fie pr 1. Tr als m Tw 45 fo is Tf iro W en in F
eld, the pub revalence for .3.1 Tran ransferrin (T so known as mediated end wo types of 5% identity i or regulating less restricte fR1; thus, it on uptake.90 When ligands ndocytosis, a nto two main
igure 7 – An ov clathrin-coated early endosome blications th r targeting m sferrin and Tf) is an iron s holo-Tf, b ocytosis.85 Tf receptors in the extrac iron metabo ed.88,89 TfR2 is believed t s, such as h as shown in F n pathways: l verall diagram o d vesicles. After es, as Tf shows n
hat deal wit medical diagn d its recepto n-binding pr inds to the s exist, TfR1 cellular doma olism such a affinity towa that TfR2 ha holo-Tf, are Fig.7, endoc ate endosom f Tf trafficking. the uncoating o no preference f th analytica nostic applica ors rotein that fa Tf receptor and TfR2.86 ain.87 TfR2 i as the liver an ards holo-Tf as a role in o e internalize cytosed ligan mes or recycl . Tf is internaliz of clathrin, Tf c for either popul
al application ations. acilitates iron (TfR) and 6 TfR2 is a cl s predomina nd small inte f is approxim overall iron m ed into the nds are sorte ing endosom
zed via clathrin-can be found in b lation. LDL, wh ns are in th n-uptake in c enters the c lose homolog antly found i estine where mately 25-fol metabolism in cell throug d at the leve mes.91 -mediated endo both population hich follows the
he minority cells. Iron-lo cell through gue of TfR1 in tissues res eas TfR1 dis ld lower com in addition t gh clathrin-m el of early en
ocytosis and tak ns of static and degradation pa y, with a oaded Tf, showing sponsible stribution mpared to o cellular mediated ndosomes ken up into dynamic athway, is
w D its of fo de su Fig by ext car A ev co Ph trafficked pref which proceeds t directly back com espite its ub s expression f the recepto or cancer cel elivery of dru ummarizes va gure 8 - Exampl y its natural liga tracellular dom rrier. Reproduc Among these valuation:93 M ompleted a P hase Ib/II cl ferentially to dy to separate from k to the plasma mpartment befo biquitous exp can be corr or on maligna ll proliferatio ugs into mal arious ways t
les of strategies and Tf, a specifi main of the TfR ced from Danie
e, it is worth MBP-426 is Phase I clin linical trial i
ynamic early end m early endosom a membrane. A s ore Tf is traffick
pression, TfR elated with t ant cells, its c on make thi
ignant cells a through whi
s for targeting th fic peptide, mon R. Therapeutic a ls et al.93 noting that a liposome nical trial (2 n second lin dosomes. Tubu mes and traffic slower recycling kedback to the c R1 is signific tumor stage constitutive is receptor a and thus, an ich the transf
herapeutic agen noclonal antibo agents can be d t two liposom e conjugated 2006, NCT0 ne patients w ulation occurs in Tf to recycling g route delivers cell surface. Rep
cantly over-e or cancer p ability to int an ideal and n attractive ta ferrin recept nts via TfR to m odies or single c delivered conjug mal formulat d with Tf fo 00355888). T with gastric, n both populatio pathways. A fas s Tf first to the p produced from M expressed on progression. T ternalize and widely acce arget for can
or was used
malignant cells. T chain antibody gated to the com
tion reached r the deliver This formul
gastroesoph
ions of early end st recycling rout perinuclear recy Mayle et al.92 n malignant This high ex d the necessit essible porta ncer therapy. in cancer th Targeting can b fragments spec ompound, or en d the stage o ry of oxalipl lation also r hageal, or es dosomes te takes Tf ycling cells and xpression ty of iron al for the Figure 8 erapy. be mediated cific for the nclosed to a
of clinical latin that eached a ophageal
adenocarcinoma (2009, NCT00964080). The other system that underwent clinical evaluation in a Phase I clinical trial is the SGT-53 immunoliposomes using antibody fragments as ligands. SGT-53 is a liposome that encapsulates plasmid DNA coding for wild type p53, forming a complex that is targeted to tumor cells by means of an anti-TfR scFv (TfRscFv) attached to the surface of the liposome (2007, NCT0040613).
1.3.2 Peptide aptamers that target the transferrin receptor
In 2001, Lee and coworkers developed two promising peptides able to target transferrin receptor.94 A biopanning process designed to find peptide epitopes specific for cell surface receptors was used in the study, to select 7- and 12-amino-acid peptides capable of binding to and internalizing with the human transferrin receptor (hTfR). Interestingly, transferrin did not compete with either of these sequences, suggesting that these peptides bind a site on the hTfR distinct from the transferrin binding site. When either of these sequences was expressed as a fusion to green fluorescent protein (GFP), the recombinant GFP molecule was internalized in cells expressing the hTfR. This work paved the way for the use of these two peptides to target other payloads into the endosomal pathway. However, the 12-aminoacid one was later found to promote aspecific uptake of its conjugates into various cell lines and as such its use was abandoned.95
Conversely, the 7-mer HAIYPRH (referred to as T7) found widespread applications in the following years. In an excellent example, this peptide was covalently conjugated to artemisinin in two different ways to form targeted conjugates and these demonstrated anti-cancer activity against Molt-4 human leukemia cells with an IC50 of about 1 μM. They were not toxic to normal lymphocytes (IC50 > 10 mM), demonstrating cancer cell selectivity of the conjugates.96 Moreover, the peptide was used in targeted PAMAM dendrimers, both as a vehicle for drug delivery97 and for targeted, contrast-enhanced MRI98 and for the potential therapy of glioma.99
2 Results and discussion
2.1 General remarks
This thesis was developed in the framework of an ongoing research line carried out at NEST laboratory, on the engineering of new contrast media for ultrasound sonography and theranostics.
An ideal, third generation contrast medium has a long circulation time in the bloodstream, can be targeted to specific cells or tissues and may be linked to a drug carrier to deliver pharmaceutical substances of interest to the aforementioned targets. Different parts of the microbubble take care of these properties and each one of these modules has to be optimized in order to achieve the best results.
The structure of a possible contrast medium, inspired by recent works,67 is depicted in the following scheme.
Scheme 1 - Scheme of the engineering approach that leads to liposome-loaded microbubbles.Liposome-loaded microbubbles (right) can be made by linking liposomes (center) onto their surfaces. Stealth and targeting properties
can be conferred to these structure through the use of lipopeptides (left).
In this work, we focused on the production of stealth, targeted and drug-loaded liposomes, used to coat the shell of these microbubbles. To this end, we carefully optimized and tested each component of these modular vesicles through several steps:
- As a target, we chose the human transferrin receptor (hTfR) and we achieved binding selectivity by incorporating a novel lipopeptide, which includes a peptide aptamer fragment, into the liposome shell.
- The stealth properties of liposomes were obtained by devising a new class of stealth lipopeptides, inspired by natural long-circulating proteins and by incorporating them into the liposome shells.
-
These liposomes were coupled to linkable microbubbles, using maleimide-thiol chemistry, yielding liposome-loaded, targeted microbubbles.2.2 Peptide and lipopeptide synthesis
2.2.1 Targeting peptides
As already mentioned in the introduction, the paper of Lee and coworkers identifies two potential peptides to be used in a targeting setting.94
The first one is a 12-residue oligopeptide (T12) with sequence THRPPMWSPVWP, which shows an excellent affinity for the human transferrin receptor (with a Kd ~ 1.5 × 10-8). The second one (T7) has seven residues, with sequence HAIYPRH and a Kd ~ 4.4 × 10-4. Notably, these peptides do not bind the same specific site of human transferrin and that the endogenous protein is required for internalization processes.94
The 12-aminoacids peptide seems to be the more appealing, owing to its high affinity for the receptor. However, as already discussed in the first chapter, a study suggested that this peptide could bind aspecifically to many serum proteins. Conversely, a review of the literature concerning peptide T7 shows that it has already been applied both in vitro and in vivo.
2.2.2 Labeling of targeting peptide T7
The development of a T7 analogue was needed in order to confirm that this peptide was able to bind and internalize in cells in our experimental conditions. Notably, use of T7 on MIA PaCa-2 cells (the ones we chose for the in vitro experiments) was not documented, thus we had to verify the ability of our peptide to perform internalization. To this end, we prepared a new peptide, T7-GC, provided with a cysteine residue suitable for bioorthogonal labeling with fluorescent probes, by means of solid phase protein synthesis (SPPS).
The synthetic procedure adopted for T7-GC needed some precautions during the work-up and general handling of this derivative, due to the free cysteine residue: first of all, the peptide was cleaved from the resin and deprotected using a different cleavage solution, which included 2% by volume of 1,2-ethanedithiol (EDT), to avoid cysteine oxidation. No cautionary measures were undertaken during the purification by HPLC, since the instrument has an inline degasser which removes oxygen and other gases from the eluents. Furthermore, it is known that cysteine oxidation is nearly abolished in acidic media, such as those used during HPLC purification. The purified product was handled under inert atmosphere or using degassed solvents to preserve the oxidation state of the cysteine.
This procedure afforded T7-GC in good yield: the HPLC analysis of the crude showed a purity of 59%, measured by the absorbance at 280 nm (Fig. 10). After HPLC purification and freeze drying, the product was recovered as a white solid, with a final yield of 28 %.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 50 100 150 200 250 300 350 T7-GC - 13.7 min Absor b ance at 280 nm (AU) Time (minutes)
Figure 9 - HPLC chromatogram for the synthesis of T7-GC
Concerning the fluorescent label, our fluorophore of choice was Atto 633. This dye has the advantages, compared with other commercially available dyes, of bearing only one positive charge and of being highly photostable with excellent brightness. Additionally, Atto 633 emission (in the far red/NIR domain) is spectrally well separated from cell autofluorescence. This allows unequivocal detection of internalized liposomes, without the risk of spectral overlaps.
Th ca re T7 ad to co In pr of Th de 2. N lin In N wa Bo Fi C in 2. H m m he coupling an be used i esidues. Follo 7-GC with dvantage (ov owards free oupling agen n agreement roceeded sm f the peptide he pure pro etermined by .2.3 T7-A Next, we evalu ne derived fr n our experim Not surprising as observed ovine Serum igure 10 - Intern incubati onversely, n ncubation, hi .2.4 Targe Having found millimolar sca molecule was reaction was in aqueous owing establ 2 equivale ver dithioth thiols. This nts can be add t with what moothly and e substrate in duct was fre y UV-VIS sp
Atto633 and uated the bin rom human p ments, T7-A gly, extensiv after short in m (FBS) (Fig nalization of T7 on with the labe
no internaliz ighlighting th eting lipop d that T7 wa ale, we tried s based on t s carried out conditions a lished protoc nts of TCE reitol and s eliminates t ded directly t is reported HPLC analy n 2 hours; pu eeze-dried, re pectroscopy. d MIA PaC nding and in pancreatic ca Atto633 was ve vesicular s ncubation tim g. 11). 7-Atto633 in MI eled peptide. Le zation took he crucial rol peptide Pep as a suitable the synthes the studies t using Atto and readily cols for the m EP (tris(2-c similar reduc the need fo to the crude d for the la ysis of the re urification o econstituted Ca-2 cells nternalization ancer. s administer signal arising mes, when c IA PaCa-2 cells. eft: Atto 633 flu
place when le of transfer p-T7 targeting pe sis of the co of Stefanick 633-maleim forms stabl maleimide-cy carboxyethyl cing agents) r purificatio e reduction m abeling of s eaction mixtu of the produc d in HEPES n properties red at 1 μM g from inter culture mediu . Scale bar: 20 μ uorescence; cen n serum-free rrin in promo eptide and th orresponding k and cowor ide, a biocom e, covalent ysteine coup l)phosphine) ) of being e on of the int mixture. similar pept ure showed c ct was easily buffer and i of T7 in MI concentrati nalization of um was supp μm. Cells were im nter: transmissio medium w oting interna
hat its synthe g lipopeptide rkers.102,103 In mpatible rea bonds with pling, we pre ).100 TCEP essentially un termediate t tides,101 the complete co y achieved by its concentra IA PaCa-2 ce ion to cultu f the labeled plemented w imaged after 30 on; right: overl
was employe alization.
esis was feas e. The desig In their wor agent that cysteine e-reduced has the nreactive thiol and reaction onversion y HPLC. ation was ells, a cell red cells. d peptide with Fetal minutes of lay d during sible on a gn of this rk, it was
showed that cellular uptake of liposomes can be significantly enhanced by tuning the hydrophilicity and the PEG content of the targeting peptide. In particular, the best results were obtained when three residues of lysine were incorporated into the lipopeptide, along with a PEG chain not heavier than 250 Da (5/6 units of ethylene glycol). This structure (Fig. 12), shows how we designed our lipopeptide based on those observations.
Figure 11 - Design of the lipopeptide Pep-T7. Two acyl chains are linked to he peptides by amide coupling with a
lysine. This residues is followed by a Eg3 spacer, three residues of lysine, and Eg2 spacer, and the targeting sequence.
The lipopeptide was assembled by standard SPPS approach, with minor modifications compared to standard protocols. The Eg2 spacer and the Eg3 linker were inserted using their respective Fmoc-amino acidic derivatives and the two palmitic acid chains were inserted using a bi-Fmoc-lysine residue.
Despite the increased structural complexity of this molecule, the SPPS allowed to synthesize the lipopeptide with acceptable purity (40% as measured by HPLC). The two major byproducts arose from the incomplete coupling of terminal palmitic acid, as shown by the chromatogram of the crude post-cleavage mixture (Fig. 14).
0 2 4 6 8 10 12 14 16 18 20 22 24 0 30 60 90 120 150 180 210 240 270 "Monopalmitic" Pep-T7 9.3 and 9.8 min Pep-T7 - 15.3 min Abs o rbance at 2 80 nm(AU) Time (minutes)
The main peak at 15.22 min was identified as the one of Pep-T7, while the two minor peaks at 9.23 min and 9.80 min are two “monopalmitic” products: that is, the two products in which the double coupling of palmitic acid with the di-Fmoc lysine was only partially successful.
No investigations were further attempted to elucidate which peak belonged to the specific “monopalmitic” product, but it can be speculated that the most abundant one (at 9.80 min) belongs to the monoderivatized product bearing a palmitate tail attached to the ε nitrogen (i.e. nitrogen on the side chain of lysine), since this is known to be more reactive than the α nitrogen. Notably, analysis of the reaction byproducts clearly evidenced that the most critical step is the final coupling of the two palmitate residues with the terminal lysine. Optimization of the coupling conditions was beyond the scope of this thesis, but it has to be underlined that it should be relatively easy to obtain the crude product in excellent purities (over 60%) by simply fine-tuning one coupling step.
2.2.5 Stealth lipopeptides
The idea of a novel stealth lipopeptide that was able to overcome the limitations of pegylated lipids described in the introduction was inspired from a study of Nowinski and coworkers.104 In their work, gold nanoparticles were coated with a stealth peptide (with sequence NH2-EKEKEKE-PPPPC-CONH2), mimicking the most abundant amino acid residues present on the surface of soluble proteins. These particles were able to withstand aggregation in undiluted human serum for 24 hours, and their nonspecific uptake from endothelial and immune cells was found to be minimal.
Thus, we decided to synthesize an analogous lipopeptide derivative, rationally designed on the basis of its localization and desired function.
While designing our lipopeptide, we took into account several factors:
1. in order to confer stealth properties to the liposomes, the lipopeptide had to retain a terminal sequence equal to that of the peptide devised by Nowinski. We decided to keep the sequence EKEKEKE-PPPP, attaching the other residues (i.e. the lysine and the palmitic tails) to the terminal proline.
2. The stealth units are meant to reduce aspecific protein adsorption, but should not hamper binding and internalization process. Thus, we partially retained the general structure of the targeting lipopeptide proposed by Stefanick, keeping three lysine residues which impart an additional hydrophilicity to the lipopeptide.
3. We decided not to add additional Eg spacers between the targeting sequence and the palmitic acid tails, since the PPPP fragment, which forms a rigid alpha-helix, is already designed with that purpose.
Figure 13 - Structure of Pep-Stealth
The synthesis of Pep-Stealth afforded the desired lipopeptide in excellent yield. The purity of the product after cleavage, measured by HPLC-DAD at 280 nm, was 67%. Its isolation was easy due to its hydrophobicity, which allowed for a good separation by preparative RP-HPLC, as shown by the following chromatogram (Fig. 15).
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 1000 2000 3000 Pep-Stealth - 19.6 min Abs o rbance (A U) Time (minutes)
2.
W as ho of M ca ste F d Fi ag dr a r vig Th m th.3 Lipos
With all the m ssembly of lip omogeneous f lipopeptide Moreover, thi an be stored eps involved Figure 15 - Sche dried lipid mixt
LUVs. (1): dry irst, a mixtur gain in a mix ried (step 1) round botto gorously agi he vesicles p membrane of he aqueous m
some synt
modules, bot posomes. Al s lipid mixtu es into liposo is method al at -20 °C un d in this proceme of the lipos ture; (c): hydrat ying step; (2) an re of the lipi xture of 99.5 ), to yield a h omed-flask an tating this m produced th f suitable por medium (f).
thesis
th lipidic and ll liposome sre. This syst ome membra
lows the use nder nitroge cedure are sh
some preparatio ted lipid mixtur nd (3): dispersi ds and lipop % cyclohexa homogenous nd dispersed mixture c for
his are finally rosity, yieldin d pepditic, i syntheses we tem proved t anes, compar er to stock fr en atmosphe hown in Fig. on procedure. ( re; (d): dispersi ion in aqueous m petides in ch ane and 0.5% s solid mixtu d in the aque 1 hour (step y downsized ng the small in our hands ere carried o to be the mo red to the “cl reeze-dried m ere for month
16.
(a): lipid mixtur ion of MLVs; (e media and agita
hloroform or % of ethanol ure of lipids ( eous medium p 3), yields a d with and e l unilamellar s, we turned out by hydrat ost efficient f lassic” thin-f mixture of lip hs, before be re in a solution e): liposome ext ation steps; (4) r methanol is (a). The so (b). The mix m of choice ( dispersion o extruder e (s r vesicles of i d our attentio tion of a free for the encap film hydratio ipids. These eing rehydra of organic solve truder; (f): disp and (5): extrus s dried and d olution is the xture is trans (step 2). Hea of MLVs (d) step 4), fitte interest, disp on to the eze-dried psulation on.105 mixtures ated. The ents; (b): persion of sion step dissolved en freeze-sferred to ating and . ed with a persed in
This procedure has several advantages compared other procedures, such as hydration of monophase solutions and reverse-phase hydration8:
- It is quick (the overall process requires less than 2 hours, including the hydration step), cheap and yields a narrow-sized distribution of liposomes.
- Freeze-dried mixture of lipids can be prepared and stored at -20 °C for an indefinite amount of time, ready for hydration.
This method can be used directly to encapsulate drugs inside the liposomes (by hydration in the presence of high concentrations of the payload). However, a low encapsulation efficiency occurs when hydrophilic drugs are used. So far, the better strategy to achieve high encapsulation efficiencies (up to over 98%) is the use of an active loading technique.106
2.3.1 Simple Liposomes
First of all we prepared simple, plain liposomes, composed exclusively of lipid components, with the aim of validating the robustness of the preparation protocol. Furthermore, their behavior was kept as a benchmark to test efficiency of encapsulation process, interaction with serum proteins and internalization in cells.
In the preparation of our first benchmark liposome (Lipo-1), we selected a phospholipid/cholesterol ratio of 4:1, since several works claim that this is the optimal composition for drug delivery.107 Lower cholesterol percentages confer a certain degree of unpredictability to the membrane properties even for small variations in lipid composition,108 while higher amounts make the membrane too rigid, hampering the drug loading processes. In order to provide a better comparison between the synthesized liposomes, this ratio was maintained for all the other: when lipopeptides or PEG-modified lipids were added to the mixture, their amount was accounted into the “phospholipids” quota and the percentage of DSPC decreased accordingly.
Preliminary evaluation of the properties of Lipo-1 showed that the liposome presents a reasonably narrow size distribution (Fig. 17).
1 10 100 1000 10000 0 10 20 Pe rcen ta ge Mean diameter (nm)
Figure 16 - Size distribution for simple liposomes "Lipo-1", as measured by DLS. Dispersant: water; T: 37 °C. Mean size: 234.4 nm. S.D.: 61.46 nm.
Subsequently, Zeta potential of this liposomal formulation was evaluated.
Zeta potential is a physical property that belongs to all particles in suspension and is closely related to the surface charge. The Zeta potential of a colloidal dispersion can be linked to its stability and evaluation of this parameter is of paramount importance when designing a new liposomal formulation. The analysis of the liposome dispersion showed a broad, monomodal peak, with a weakly negative charge (-3.47 ± 14.5 mV).
This nearly neutral value is quite reasonable, owing to the presence of natural zwitterionic lipids on the shell. We can assume that the slightly negative potential is related to the presence, in Lipo-1 as well as in the other liposomes, of a small percentage (0.5 %) of rhodamine-labeled lipid (DOPE-Rhod). Indeed, this lipid bears a negative net charge owing to the derivatization of the amino group with the dye, and could marginally contribute to the net surface charge of the liposome.
Unfortunately, neutral liposomes prepared with this approach showed a remarkable tendency to aggregate. Indeed, significant degradation of these liposomes was observed even upon short-term storage (7 days) in controlled conditions (8 °C, in the dark). As shown in Fig. 18, size distribution mesaured by DLS evidenced the presence of aggregates with a mean diamater of several microns, along with remarkable broadening of their size distribution (282.8 ± 125.6 nm).
1 10 100 1000 10000 0 5 10 Per cen tage Mean diameter (nm)
Figure 17 - Size distribution of liposomes "Lipo-1" after 1 week of storage, as measured by DLS. Dispersant: water; T: 37 °C. Mean size: 282.8 nm S.D.: 125.6 nm
This observation is in agreement with what is reported in the literature by taking into account the close-to-zero surface charge of the liposome membranes: these so-called “neutral” liposomes have low stability in aqueous media and, even in the best conditions, do not last longer than a few weeks.109
In order to prepare more stable liposomal dispersions, we turned our attention to the formulation of simple, non-functionalized negative liposomes that, according to the literature, are significantly more stable than neutral or quasi-neutral ones. This second batch, “Lipo-2”, was prepared with the addition of a small percentage (5%) of the anionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), which is frequently used to this end. Fig. 19 shows the size distribution of Lipo-2 after synthesis.
1 10 100 1000 10000 0 10 20 Perc entage Mean diameter (nm)
Figure 18 - Size distribution for simple liposomes with DPPG "Lipo-2”, as measured by DLS. Dispersant: water, T: 37 °C. Mean size: 206.4 nm. S.D.: 59.98 nm.
The surface Zeta potential of these liposomes was -18.7 ± 7.4. It is widely accepted that a Zeta potential whose absolute value is greater than 30 mV has is a good indicator of long-term stability, although formulations with Zeta potential as low as 15 mV have been shown to be stable for months, moreover, sterically stabilized particles (such as pegylated liposomes) are kinetically stable even with a lower zeta potential.110
In accord to the predicted increased stability of negative liposomes, analysis of these liposomes one month after their extrusion (stored at 8 °C), showed only a minimal broadening of their distribution (5%), which reached 215.6 ± 63.4 nm, in agreement with their predicted stability.
To test the efficiency of the encapsulation process, the liposomes were hydrated with a 140 mM solution of ammonium hydrogen phosphate (NH4)2HPO4. The extraliposomal solution was exchanged twice with HEPES by membrane dialysis to set up the gradient for encapsulation.
While the authors of this procedure reported that DOX encapsulation was possible even at low temperatures (7 °C),106 in our hands the drug was not able to enter the intraliposomal space in these conditions. However, heating the dispersion to 60 °C for 60 minutes allowed for an almost complete encapsulation of the drug (97 %).
Indeed, the difficult loading of DOX into liposome at low temperature could be reasonably correlated with the rigid behavior of Lipo-2 membrane at low temperature (Tc of DSPC and DPPG are 55 °C and 41 °C, respectively).111 Conversely, the second set of conditions, where the dispersion is heated above the transition temperature of the phospholipids, is the most common one for the gradient-driven loading procedure, such as those that employ an intraliposomal environment rich in ammonium sulfate.112,113
2.3.2 Stealth Liposomes
As already discussed in the introduction, one of the main drawbacks of conventional liposomes last is their short half-life in the bloodstream; indeed, adsorption of serum proteins on their surface (opsonization) occurs within a few minutes, leading to aggregates that are readily recognized by the reticuloendothelial system (mainly phagocytes). The so-called stealth liposomes, which include pegylated lipids, proved to be the most successful for to overcome this issue,31,4 but as showed in the introduction, they have their drawbacks too.
With the aim of producing a new class of stealth liposomes, we evaluated the influence of different percentages of stealth lipopeptide Pep-Stealth on liposome behavior and stability. In order to evaluate the best performing composition, the formulations tested in this preliminary screening included either 100%, 50% or 25% of Pep-Stealth (not taking into account the cholesterol quota) and DSPC.
