WO2014071072A2

WO2014071072A2 - Novel cationic carotenoid-based lipids for cellular nucleic acid uptake

Info

Publication number: WO2014071072A2
Application number: PCT/US2013/067869
Authority: WO
Inventors: Michael D. PUNGENTE; Vassilla PARTALI; Hans-Richard SLIWKA; Christer L. OPSTAD; Phillip L. LEOPOLD; Howard H. LOU; Natalia BILCHUK; Emile JUBELL
Original assignee: Pungente Michael D; Partali Vassilla; Sliwka Hans-Richard; Opstad Christer L; Leopold Phillip L; Lou Howard H; Bilchuk Natalia; Jubell Emile
Priority date: 2012-11-02
Filing date: 2013-10-31
Publication date: 2014-05-08
Also published as: WO2014071072A3

Abstract

The synthesis and self-assembling properties of a model compound in a new class of cationic phospholipids with a highly unsaturated conjugated carotenoid fatty acid are described. In addition, the potential of this new lipid as a nucleic acid carrier was evaluated through lipoplex formulations employing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as helper lipid with and without the polycationic peptide, protamine, together with a plasmid DNA (pDNA). Lipoplexes composed of this novel unsaturated lipid exhibited pDNA binding and protection from DNase I degradation when formulated with protamine. Cellular uptake of the liposomes could be monitored by the visible color of the carotenoid moieties. The new cationic lipid gene delivery vector revealed comparable transfection efficiency to the commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphophocholine (EPC), in Chinese hamster ovary-K1 (CHO-K1) cells and performed equally to Lipofectamine 2000 when the formulation included protamine.

Description

NOVEL CATIONIC CAROTENOID-BASED LIPIDS FOR CELLULAR

NUCLEIC ACID UPTAKE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Ser. No. 61/721,975, filed

Nov. 2, 2012, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND

Cationic lipids are promising non- viral vector systems for use in small interfering RNA (siRNA) and DNA delivery to effect the introduction of exogenous sequences of DNA into cells to correct defective genes or, to selectively silence gene expression, referred to as RNA interference, or RNAi, through siRNA delivery. Over the past several years, novel cationic lipid vectors have been synthesized and combined with nucleic acids for these purposes.

In order for the lipid-nucleic acid complex (or lipoplex) to cross the cell membrane, the complex should be charge- neutral or have an excess positive charged overall. The use of cationic lipids facilitates lipoplex formation by developing a charge-neutral complex with the negatively charged nucleic acid (DNA or siRNA). Although unsolved, the mechanism by which the lipid/nucleic acid complex is internalized into the cell is thought to occur by endocytosis. The lipoplex size is important for active endocytosis. Lipoplexes greater than 250 nm in diameter resulted in larger endosomes that are more easily ruptured. Larger particles may have better contact with the cells increased phagocytic activity accompanied by endosomal escape. It should be mentioned that large particles on the order of the size of a cell are inefficient for in vivo

administration as they become trapped in the capillary regions.

In the case of siRNA delivery, the lipoplex must escape the endosome and traffic the cytoplasm where the siRNA is taken up by the RNA-induced silencing complex (RISC), leading ultimately to the catalytic destruction of a complimentary endogenous messenger RNA (mRNA). This results in preventing the native mRNA from producing a protein product; this process is referred to as "knockdown". However, knockdown is not without restrictions when it comes to practical applications. The clinical application of RNAi is restrained by lack of tissue specificity, degradation of the complex by cellular components, and toxicity associated with the cationic lipid carrier.

Current commercially available cationic glycerolipids used for siRNA delivery are not effective enough as siRNA delivery vectors. These lipids are characterized by a common structural motif that includes a hydrophilic headgroup, linker bond, backbone (typically glycerol) and two hydrophobic tails, mainly as saturated fatty acid chains.

Nucleic acid binding by the cationic lipid vector calls for a headgroup that can sustain a positive charge at physiological pH. To achieve this, typical headgroup moieties include primary, secondary, or tertiary amines, and in addition, quaternary ammonium salts, guanidine, and imidazole groups have been successfully employed. A large number of cationic lipids are functionalized with polyamine headgroups, where spermine and spermidine groups are very common. Some reports suggest that since formulations using polyvalent lipids require a lower stoichiometric amount of cationic lipid to reach charge neutrality with the negative charges associated with the phosphate groups of the nucleic acid, such polyvalent lipids are typically less toxic. A risk associated with the polyvalent cationic headgroup is that the electrostatic interaction between the lipid and nucleic acid cargo is too intense, resulting in failure to release the cargo to allow of the intended function of the genetic material. Non- viral lipid vectors functionalized with the quaternary amine headgroup are reported to be more toxic than those containing the tertiary amine headgroup.

Cationic lipids have been used as nucleic acid carriers to eukaryotic cells for 25 years. ^[1] Nevertheless, gene transport with these lipids is still in a trial and error phase, as illustrated in a study wherein 1200 tested compounds revealed that only 65 (5 %) delivered nucleic acid into cells as well as or better than Lipofectamine^[-^], a commercially available gene delivery formulation. ^[-^] The many biological and chemical variables have so far prevented establishing an unambiguous structure- activity relationship. ^[—^] Even so, about 6% of all clinical gene therapy trials are based on lipid/nucleic acid complexes

(lipoplexes) prepared with cationic phospholipids. ^[-^] Most glycerolipidic gene carriers contain flexible saturated or low unsaturated chains. Rigidity has been introduced with steroids, diphenylethyne and triazine dendrimers.^[-' -' -^] To our knowledge, no precedence for the interplay of polyunsaturated- stiff and saturated-flexible chains in nucleic acid carrying phospholipids has yet been specified -¹ although this association may be important for adjusting morphology changes along with phase state transitions (cylindrical shape ^→ cone shape = lamellar phase^→ inverted hexagonal phase). ^[—^] Hexagonal phases are either contradictorily considered important or irrelevant for gene transfer. ^{[li ]}

SUMMARY

The invention, in various embodiments, is directed to a carotenoid lipid of formula (I)

Car-C(=0)-L-Cat(+) X(-)

(I)

wherein Car is a carotenoid moiety, C(=0) is a carbonyl group bonded to a terminal ethenyl group of the carotenoid moiety, L is a linker bonded via an ester bond to the C(=0), linker, L optionally comprising a phosphate (Cl-C30)alkyl ester, Cat can be a cationic quaternary ammonium, and X is an anion; to methods of preparation of compounds of formula (I), and to methods of use of compounds of formula (I) in liposome-mediated transfection of cells with exogenous nucleic acids such as DNA and small interfering RNA (siRNA).

For example, the invention can provide a carotenoid lipid wherein L is a group comprising a glycol phosphate ester group, the lipid being a carotenoid glycol p

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Ci- C₄)hydroxyalkyl, R⁴ is (Ci-C3o)alkyl, ml is 1, 2, or 3, m2 is 1, 2, or 3, and n is 1,

2, or 3 ; X is an anion.

For example, the invention can provide a carotenoid lipid wherein L is a chain comprising one, two, or three (C2-C4)oxyalkylene group bonded via an oxygen atom to the carbonyl group, the lipid being a carotenoid glycol lipid having formula (IV)

(IV)

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Q- C4)hydroxyalkyl, nl is 1, 2, or 3; and, n2 is 1, 2, or 3; X is an anion.

For example, the invention can provide a carotenoid glyceryl phospho

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Ci- C4)hydroxyalkyl, R⁴ and R⁵ are each independently selected (Ci-C3o)alkyl, and n is 1, 2, or 3; X is an anion.

termed herein a "C-20 carotenoid chain", i.e., C-20 including the carbonyl group of formula (I), wherein a wavy line indicates a point of bonding to the carbonyl group of the carotenoid lipid of formula (I). The cationic group Cat can be a choline group (-Οί₂0¾Ν(0¼)₂), or can be an analogous quaternary ammonium group, or an ammonium salt of a primary, secondary, or tertiary amine group.

Anion X, a counterion to the cationic group Cat, can be a

pharmaceutically acceptable anion, such as bromide or iodide.

The invention can further provide a liposome, such as a

dioleoylphosphatidyl-ethanolamine (DOPE) liposome, comprising a carotenoid lipid of the invention. The liposome of can further comprise a polynucleotide, such as a si(RNA) or a DNA, contained therein.

In various embodiments, the invention can provide a method of transferring a polynucleotide to the interior of a living cell, comprising contacting the cell and an effective amount or concentration of the liposome comprising a carotenoid lipid of the invention, and containing the

polynucleotide, such as DNA or siRNA. In various embodiments, an immune response is not induced by the contacting of the liposome and the cell within the living organism. In various embodiments, the polynucleotide is incorporated into the genome of the cell, or interferes with expression of the genome of the cell, or both.

The novel cationic carotenoid-based lipids within are inherently colored, and once they form an electrostatic complex with negatively charged nucleic acids (DNA or siRNA), the colored complex can be tracked in experimental samples including cultured cells (in vitro) or experimental animals (in vivo). The C20- or C30-carotenoid chain associated with these novel lipids introduces color to the gene carriers, providing confident handling of the lipid throughout formulation as well as instant visual confirmation of treated versus non-treated cell cultures. The utility of this property was demonstrated by detection of the transfection reagent macroscopically in the dermis of nude mice at the site of lipoplex injection, and microscopically in intact cells. By combining a C20- or C30-carotenoid lipid with a more effective gene transfer reagent, for example EPC, the benefits of both compounds were realized in a single formulation.

In various embodiments, the invention provides a method of treatment of a medical disorder in a patient wherein administration of a liposome containing a polynucleotide is medically indicated, comprising administering to the patient an effective amount of the liposome of the invention. For example, the medical disorder can be Duchenne muscular dystrophy.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows C20-n and C30-n cationic phosphocholine lipids composed of a glycol scaffold, a hydrophobic polyenoic chromophore (C20:5, C30:9), hydrophopic alkyl chains and a hydrophilic head group. The designation n describes the length of the alkyl chain of the phosphate ester, group R⁴ of formula (III).

Figure 2 depicts the structure of dioleoyl (Ci8:i) zwitterionic ( ?)-DOPE, cationic reference ( ?)-EPC and cholesterol.

Figure 3 shows hypothetical structures of rigid polyene chains and flexible saturated alkyl chains for compound C30-14.

Figure 4 depicts maximum and minimum reduction of water surface tension (γ = 73 mN/m) with surface active C20-n and C30-n compounds.

Figure 5 depicts the molecule area a_m at the water surface of C30-16 (representing roughly the average of most C20-n and C30-n molecules), of C20- 14 and C30-14.

Figure 6 depicts hypothetically oriented C30-12 (closed V) and C30-14 (stretched V) at the water surface, the solid sections of the line indicate the diameter of the surface area a_m at the water surface for the two compounds (semi-empirical, AMI in Spartan 08, Wavefunction, Irvine, California, USA).

Figure 7 shows the molecular volume (A³) of CX-n compounds and EPC. C30-12 and EPC have comparable molecular volumes (spartan08, semi empirical PM3).

Figure 8 shows results of gel retardation assays of C20-n/DNA (A) and C30-n/DNA (B) lipoplex formulations compared to EPC/ DNA lipoplexes (all without added co-lipid) at various N/P (+/-) molar charge ratios, ranging from 0.5: 1 to 10:1 , run through a 1% agarose gel impregnated with the DNA gel stain, ethidium bromide. Lanes L and D denote lanes containing a 1 kb DNA ladder or DNA alone, respectively.

Figure 9 shows the results of studies concerning gene transfer for C20-n and C30-n series. CHO-K1 cells were seeded at a concentration of 10,000 cells/well in a 96-well plate and transfected with 400 ng plasmid DNA encoding β-galactosidase enzyme using formulations containing C30-12, C30-14, C30-16, C30-18, C30-20, and EPC (without co-lipid) as described in the Methods, β- galactosidase activity was determined after 48 h using and was normalized for protein content. Data indicate β-galactosidase activity in relative light units (RLU) per mg of total protein in each sample. Each transfection experiment was performed in triplicate and an average of three independent experiments is shown (with the exception of n=9 for C30-12 to C30-18).

Figure 10 depicts data relating to cytotoxicity for C20-n and C30-n series. CHO-K1 cells were seeded at a concentration of 10,000 cells/well in a 96- well plate and transfected with 400 ng plasmid DNA encoding β- galactosidase enzyme using formulations containing C30-12, C30-14, C30-16, C30-18, C30-20, and EPC (without co-lipid) as described in the Methods.

Lipoplex cytotoxicity was determined after 48 hours. Each cytotoxicity experiment was performed in triplicate and an average of three independent experiments is shown (with the exception of n=9 for C30-12 to C30-18).

Figure 11 shows results of S AXS experiments for C20-20 (A) and C30- 20 (B) lipid/DNA lipoplex formulations without co lipid at (+/-) molar charge ratio 1.5:1. (Abscissa: modulus of the scattering vector. Ordinate: intensity in arbitrary units.).

Figure 12 shows structures of cationic carotenoid lipids C30-20 and C20-

20, together with commercially available cationic lipid EPC, and neutral co- lipid, cholesterol.

Figure 13 shows results of a gel retardation assay of EPC/Chol/PMO AO,

C20-20/Chol/PMO AO and C30-20/Chol/PMO AO at (+/-) molar charge ratios 0.1:1 up to 20:1; low molecular weight marker run through a 3% TBE-agarose gel impregnated with the DNA gel stain, ethidium bromide. The gel was visualized using a Geliance transilluminator.

Figure 14 shows results of a qualitative cell viability assessment by light microscopy of hSkMCs transfected with lipoplex formulations C20- 20/Chol/PMO AO (A-D) and C30-20/Chol/PMO AO (E-H) at (+/-) molar charge ratios of 0.05:1 (A, E), 0.1 :1 (B, F) 0.25:1 (C, G) and 0.5:1 (D, H) at 24 h.

Figure 15: (A)-(C) Comparison of efficiency of various lipid/PMO AO complexes (each performed in triplicate) to induce skipping of exon 45 in RNA from hSkMCs. Nested RT-PCR was performed on 300 ng from hSkMCs treated with lipoplex formulations, EPC/Chol/PMO (A), C20-20/Chol/PMO (B) and C30-20/Chol/PMO (C) at 250 nmol/L at the ratios indicated. The obtained products were separated by agarose gel (1.5%) electrophoresis against Hyper ladder IV. The full-length product (exons 44-48) is 657 bp and the skipped product (exons 44, 46-48) is 481 bp in size. Non-transfected controls are shown in D.

Figure 16: RT-PCR amplification of ribosomal 18s housekeeping gene to assess comparative RNA quality between samples transfected with various lipid/PMO AO complexes. 100 ng of RNA harvested from hSkMCs treated with lipoplex formulations, EPC/Chol/PMO, C20-20/Chol PMO and C30- 20/Chol/PMO at 250 nmol L at the ratios indicated was subjected to RT-PCR amplification. The obtained products were separated by agarose gel (2.5%) electrophoresis against Hyper ladder V. The expected product is around 130 bp in size.

Figure 17 shows dose-response comparison of lipoplex formulations (EPC/Chol/PMO, C30-20/Chol/PMO and C20-20/Chol/PMO at 250 nmol/L) to produce exon 45 skipping in hSkMCs.

Figure 18 is a schematic of delivery of lipid-siRNA complex leading to cleavage of mRNA.

Figure 19 shows that liposome and lipid-siRNA lipoplex particle sizes were determined by dynamic light scattering at 25 °C with a detection angle of 90°. Separate hydrated liposome solutions (from sterile water) composed of cationic lipid / DOPE (3 :2 mole/mole ratio) were generated in duplicate for each carotenoid lipid, 1-5, as well as for control lipids EPC and DC-Choi, and each sample was analyzed in triplicate. The corresponding liposome-siRNA lipoplexes were prepared in OPTI-MEM^® buffer at (+/-) molar charge ratios 2.5, 5 and 10. All data are the mean + standard error (S.E.) of 3 measurements for 2 different batches.

Figure 20: Luciferase knockdown for carotenoid lipoplex formulations,

1-5, and controls DC-Choi and EPC, 48 h after transfection with various N/P (+/-) molar charge ratios. Data are expressed as relative light units (RLU)^g of protein. Data are the average of three experiments, each performed in triplicates (n=9). Data are expressed as mean + S.E. *p < 0.05, **p < 0.01 for GL2 siRNA versus control non treated cells (Student's t test).

Figure 21 : Cytotoxicity of carotenoid lipoplex formulations, 1-5, and control lipids

DC-Chol and EPC, 48 h after transfection with various N/P (+/-) molar charge ratios. The percentage of viable cells was calculated as the absorbance ratio of treated to untreated cells. Data for (+/-) molar charge ratios 2.5-10 are the average of three experiments

(n = 9); Data are expressed as mean + S.E.

DETAILED DESCRIPTION

We have designed and synthesized new achiral amphiphilic glycol lipids paired with polyene and saturated chains (Fig. 1). The synthesis of the new phospholipids is based on previous connections of polyenes with phosphate groups. We investigated the surface properties of these polyene compounds, their self-assembly to liposomes and to mixed liposomes with 1,2- dioleoyl-sra-glycero-3-phosphoethanolamine (DOPE) and cholesterol (Fig. 2), we studied the lipoplex formation by combining DNA with the liposomes, and we measured the size of liposomes and lipoplexes and resolved the membrane assembly of the lipoplexes. The physical and biological data were related to the molecular structure and properties, DNA-transfection and cytotoxicity associated with these novel polyunsaturated phospholipids.

Cationic phospholipids were synthesized introducing chromophoric, rigid polyenoic C₂o:5 (C20) and C30: (C30) chains next to saturated flexible alkyl chains of variable lengths C6-₂o_:o Surface properties and liposome formation of the amphiphilic compounds were determined, the properties and structure of lipid/DNA complexes, including analysis by small-angle X-ray scattering (SAXS) was established, and DNA transfer and cytotoxicity of the lipoplexes were examined following DNA delivery to Chinese hamster ovary (CHO-K1) cells.

Accordingly, in various embodiments, the invention provides a carotenoid lipid of formula (I)

Car-C(=0)-L-Cat(+) X(-)

(I) wherein Car is a carotenoid moiety, C(=0) is a carbonyl group bonded to a terminal ethenyl group of the carotenoid moiety, L is a linker bonded via an ester bond to the C(=0), linker, L optionally comprising a phosphate (Cl- C30)alkyl ester, Cat is a cationic quaternary ammonium, and X is an anion. For example, X can be bromide or iodide. More specifically, for the carotenoid lipid of

(ΠΑ), or of formula (IIB)

wherein a wavy line indicates a point of bonding to the carbonyl group of formula (I).

For example, the invention can provide a carotenoid lipid of forumula (I) wherein L is a group comprising a glycol phosphate ester group, the lipid being a

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (d- C₄)hydroxyalkyl, R⁴ is (Ci-C3o)alkyl, ml is 1, 2, or 3, m2 is 1, 2, or 3, and n is 1, 2, or 3. For example, each of R¹, R², and R³ can independently be H, methyl, or hydroxyethyl. The group Car can be of formula (IIA) or (IIB), above. In various embodiments of the compound of formula (III),

n = 1, or ml is 1, or m2 is 1, or any combination thereof. The phosphate ester group R⁴ can be an n-alkyl chain; for example, R⁴ can be a C₂o alkyl group, such as an n-C₂o alkyl group.

Specific embodiments of compounds of formula (ΠΙ) include any one of

The invention can further provide a carotenoid lipid of formula (I) wherein L is a chain comprising one, two, or three (C2-C4)oxyalkylene units bonded via an oxygen atom to the carbonyl group, the lipid being a carotenoid glycol lipid having formula (IV)

(IV)

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Q- C4)hydroxyalkyl, nl is 1 , 2, or 3; and, n2 is 1, 2, or 3. For example, each of R¹, R², and R³ can independently be H, methyl, or hydroxyethyl. More specifically, nl = 1, n2 = 1, or both. Specifically, the

In various embodiments, the invention provides a carotenoid glyi phospholipid of formula (V)

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Q- C4)hydroxyalkyl, R⁴ and R⁵ are each independently selected (Ci-C3o)alkyl, and n is 1, 2, or 3; X is an anion. For example, each of R¹, R², and R³ can

independently be H, methyl, or hydroxyethyl. The group Car can be of formula (ΠΑ) or (IIB), above. In various embodiments of the compound of formula (V), n = 1. The phosphate ester group R⁴ can be an n-alkyl chain; for example, R⁴ can be a C₂o alkyl group, such as an n-C₂o alkyl group. The acyl group R⁵ can be an n-alkyl chain; for example, R⁵ can be a C₂o alkyl group, such as an n-C₂o alkyl

The invention can provide a liposome comprising one or more embodiments of a compound of formula (I). Liposomes, as are well known in the art, are hollow spheroidal assemblies of lipids wherein non-polar groups associate to form a lipid bilayer in a spheroidal configuration, wherein the polar groups are presented outwards towards the aqueous environment and inwards towards the hollow liposome interior which can contain water with substances dissolved therein. As described in more detail below, liposomes of the invention comprising a carotenoid lipid of the invention possess visible coloration due to the poly-unsaturated carotenoid moiety. This color can be used to track the incorporation of liposomes comprising the carotenoid into living cells or tissues, either by visual (e.g., microscopic) inspection or by spectrophotometric means.

Accordingly, the invention can provide a liposome comprising a carotenoid lipid of the invention. For example, the liposome can be a liposome formed of dioleoylphosphatidylethanolamine (DOPE) that also incorporates the carotenoid lipids, rendering them visible when incorporated into a tissue.

As described above, it is known that liposomes can serve as vectors for transfection of living cells with nucleic acids such as DNA or RNA, e.g., siRNA (small interfering RNA) that can suppress gene expression. Thus, the invention further provides a liposome of the invention further comprising a polynucleotide contained therein, such as a si(RNA) or a DNA contained therein.

Despite the availability of two families of molecules, e.g., of formulas (III) and (IV), that structurally varied in a regular manner with increasing length of the flexible alkyl chain, the physical properties of the lipids did not exhibit a simple consistent structure/property relationship with the regular changes in flexible alkyl chain length. A similarly complex biological structure activity correlation was observed with respect to gene delivery (transfection of living cells with nucleic acids). In regard to gene transfer, subtle trends were observed among family members. The heterogeneity of physical properties observed among family members combined with the complex, multiple steps in DNA delivery to cells may be responsible for the complexity of the overall structure activity relationships. However, the data set clearly established one member of the group, a C30-n compound with a saturated C₂o side chain (designated throughout as C30-20) as a viable vector with regard to gene transfer, and possessing reduced cytotoxicity relative to known cationic lipids.

Thus, the invention can provide a method of transferring a

polynucleotide to the interior of a living cell, comprising contacting the cell and an effective amount or concentration of the liposome of the invention containing a nucleic acid, such as a si(RNA) or a DNA, such as when the living cell is within a living organism, e.g., a human patient. The liposome of the invention comprising a nucleic acid contained within the hollow shell can offer the property that an immune response is not induced by the contacting of the liposome and the cell within the living organism. In various embodiments, the polynucleotide can be incorporated into the genome of the cell, or can interfere with expression of the genome of the cell, or both.

The novel cationic carotenoid-based lipids within are inherently colored, and once they form an electrostatic complex with negatively charged nucleic acids (DNA or siRNA), the colored complex can be tracked in experimental samples including cultured cells (in vitro) or experimental animals (in vivo). The C30-carotenoid chain associated with these novel lipids introduces color to the gene carriers, providing confident handling of the lipid throughout formulation as well as instant visual confirmation of treated versus non-treated cell cultures. The utility of this property was demonstrated by detection of the transfection reagent macroscopically in the dermis of nude mice at the site of lipoplex injection, and microscopically in intact cells. By combining a C30-carotenoid lipid with a more effective gene transfer reagent, for example EPC, the benefits of both compounds were realized in a single formulation.

Accordingly, the invention can provide a method of monitoring the uptake of the liposome of the invention into a living cell, comprising observing an absorbance of visible light by the carotenoid moiety Car. For example, the observation can be made by the human eye, such as through a microscope. Cells incorporating the liposome containing the colored carotenoid lipids can be seen as being of a yellow or orange color. Or, the degree of incorporation of a liposome of the invention into a living cell can be determined using

spectrophotometric methods, either micro-spectrophotometry as through a microscope system, or through cell fractionation and extraction techniques using a standard UV/visible spectrometer to measure color intensity such as at the I_mx of the carotenoid group.

Cationic lipids are often identified with phospholipids and phospholipids are habitually recognized as glycerophospholipids.^[— '— ^] Yet, the glycerol scaffold complicates structure- activity relationship investigations by possible formation of mono and di-glycerol isomers and enantiomers, and by inter- and intra-molecular acyl migration. ^[— '— ^] We, therefore, replaced the glycerol (propanetriol) backbone with glycol such as ethylene glycol; we note that glycolphospholipids occur in minor amounts in natural lipids. ^[—^] For longer glycol linker chains in this position, and/or for glycol linker chains derived from propylene or butylene glycol, appropriate reagents can be selected by the person of ordinary skill from the multitude of commercially available glycols and polyglycols. Thus, for compounds of formulas (III) and (IV), the glycol unit bonding the cationic group to the phosphate group (formula (III) or directly as a carotenoid ester (formula (IV), can be monomeric, dimeric, or trimeric, and the repeating unit can be an ethylene glycol, a propylene glycol, or a butylene glycol unit.

For the phospholipids of formula (III) we replaced one of the flexible saturated or low unsaturated acyl chains^[— ^] typically located in lipid gene carriers^[— '— ^] with -apo-8'-carotenoic acid (C30: ) (compound 2 of Scheme 1) (C30-acid, food color E160f) or retinoic acid (C₂o:5) (compound 9 of Scheme 1). Then, for the compounds of formula (III), keeping the glycol unit, the polyene chains and the charged head group constant, the length of the phosphate ester alkyl chain was varied (C₆, C₁₂, C₁₄, Ci₆, C₁₈, C₂o); we obtained two achiral C20- n and C30-n series (Fig. 1, Scheme 1). Cationic phospholipids bearing different chains or chains with conjugated unsaturation have rarely been examined. ^[— '— — ^] The dependence of transfection efficiency on phosphate ester alkyl chain length was then examined.

To prepare compounds of formula (III), using the compound designations as shown in Scheme 1 , below, reacting chlorodioxaphospholane (3) with glycol (1) (or the glycol selected to obtain a variant of this linker group L) and subsequent addition of the appropriate alcohol C_n-OH and Br₂ gave intermediate phosphate 4-n,^[— ^] which with C30-acid 2 and the coupling reagents

chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP),^[— ^] diisopropylethylamine (DIEA) and 4-dimethylaminopyridine (DMAP) formed bromophosphotriester 5-n. Aminolysis of 5-n gave C30-6, C30-12, C30-14 and C30-20 (Scheme 1). Phosphorous diesters were retrieved as byproducts and bromine reacted promiscuously with phosphorous and the exo- and endocyclic methylene groups next to the P-O-bonds in phospholane 3.^[— ^] Otherwise, trichlorophosphate (6) was reacted with bromoethanol (7) to bromophosphate 8, which with glycol (1) and alcohol C_n-OH gave again intermediate 4-n. By these sequences C30-16 and C30-18 were obtained. Similarly, phosphate 4-n reacted with retinoic acid 9 to the C20-n compounds.

The synthetic route depicted in Scheme 1 can be adapted by the person of ordinary skill to prepare compounds of formula (III) across the scope as defined. For example, not only can the linker group L be selected by choice of the appropriate glycol or polyglycol starting material, but the phosphate ester group R⁴ can be selected by the choice of the appropriate alcohol C_n-OH of Scheme 1 , as can the appropriate carotenoid Car group, examples of which are shown as compounds 2 and 9 of Scheme 1. The groups R¹, R², and R³ of formula (III) can be selected by use of amines other than trimethyl amine in the reactions of intermediates 5-n and 10-n of Scheme 1 , below.

The combination of rigidity and flexibility in the new lipids is exemplary demonstrated for C30-14 (Fig. 3). Diverse conformational arrangements of acyl chains have been previously noticed in a zwitterionic phospholipid with a hexatriene chain. ^[—^]

Scheme 1 : Synthesis of Carotenoid Glycol Phospholipids of Formula (III)

C20-n, n = s, u, is. ie, 20

C30-n, n = 6. 12_: 14, m ia. 2o

The surface tension y of the synthesized amphiphilic phospholipids was determined with a tensiometer (Pt-plate). Calculation of the tensio metric data assessed the critical aggregation concentration CM, the area per molecule at the filled monolayer a_m and other associated data such as °^M , surface

AG⁰

concentration Γ, free energy of aggregation ^a8 and adsorption surfactant performance indicator AMER, ^J equilibrium constants for aggregation and adsorption k_ag and k_a (Table 1, below). ^[— '— ^] The surface area fl_m was determined assuming no dissociation of the molecules in water (one species) or complete dissociation (two species). The values for a_m supposing complete dissociation were too high and in variance with molecular calculations. Therefore, the cationic molecules are believed to aggregate with tightly attached counter ions. The importance of tensiometric data for controlling gene transfer has been previously demonstrated by investigating Langmuir-Blodgett films. ^[—^] C20-16 is most surface active, decreasing the surface tension γ of water to 35 mN/m, whereas C20-20 and C30-14 are not effective surfactants with γ = 62 and 63 mN/m, respectively (Table 1, Fig. 4). C20-16 aggregates in water at low concentrations (CM = 10.6 μΜ), C20-20 is with CM = 75 μΜ much more soluble. The fact that CM of most surfactants decreases linearly with the number of chain carbon atoms is not reproduced with the C20-n and C30-n

compounds. ^[—'— ^] The surface area a_m is rather similar, except for C20-14 and C30-14 with unexpected large a_m values (Fig. 5).

Table 1. Surface property data of C20-n and C30-n.

μΜ mN/m μιηοΐ/ιη² A²

C20-6 31.7 46 2.05 81

C20-14 50 57 1.2 139

C20-16 10.6 35 2.32 72

C20-18 13 48 2.63 63

C20-20 75 63 2.6 63

C30-2 23.3 49 2.6 64

C30-6 19 53 2.97 56

C30-12 46.6 53 1.93 86

C30-14 41.7 62 0.87 191

C30-16 46 49 2.95 56

C30-18 16 42 2.61 64

C30-20 18.9 47 2.1 79

The hydrophobic chains in the C30-n series can adopt a "closed V" or a "stretched V"-shaped-conformation upon rotation about the oxygen-carbon bond of the phosphate ester acyl side chain (Fig. 6). The reasons why C20-14 and C30-14 orient differently are not evident. Comparable large surface areas have been detected with polyene bolaamphiphiles.^[— ^] The C20-n and C30-n molecules with rigid and flexible chains are not directly comparable with the phosphate O-ethyl esterified phospholipid, O-ethyl-dioleoylphosphatidylcholine (EPC) containing monounsaturated chains. The comparison of the calculated molecular volumes, which are independent of chain constitution and orientation, revealed that EPC is equal to C30-12 (Fig. 7).

Aggregation of the C20-n and C30-n phospholipids started at irregular concentrations CM (Table 1) and the shape of the formed liposomes was hardly foreseeable, e.g. resulting for C30-2 in rods, spheroids and cones. ^[—^] It is assumed that the other C20-n and C30-n carotenoid lipids similarly self- assemble in water to polymorphic particles. Independent of their morphology, aggregates are characterized by their absorption spectrum reflecting the molecular arrangement in the aggregation unit,^[— ^] e.g. the aqueous aggregate dispersion of C30-14 absorbed at lower wavelengths than a molecular solution in organic solvents, suggesting an exitonic card-pack association of the molecules characterized as H-aggregates (C30-14 in EtOH l_max = 444 nm, C30- 14 in H₂0 (H-aggregate) l_max = 400 nm.^[— '— ^] Adding water to concentrated solvent solutions of the C30-n lipids allowed detecting the aggregation concentration c_ag correspondingly, adding organic solvents to aqueous C30-n dispersions assessed the concentration of aggregate disruption, see Table 2. The monomer- aggregate equilibrium for C30-14 was, for instance, found at 31% EtOH and 69% H₂0.

Table 2: Aggregation concentration of cationic lipid C30-2 (% H?Q in solvent).

MeOH EtOH i-PrOH MeCN

67 72 77 81

Hydration of C20-n and C30-n films gave 2 mM dispersions of lipids in the form of liposomes, whose particle size was ascertained by dynamic light scattering (DLS). C20-18 liposomes revealed the largest hydrodynamic diameter (dn = 1645 nm), however, the high polydispersity index (Pdl = 0.8) indicated a mean value from particles of varying sizes. Cholesterol and zwitterionic DOPE

(nucleic acid protectors and transfection promoters) were included as neutral co- lipids and paired with lipids C20-n and C30-n at a constant molar ratio of 3 :2

(cationic lipid/co-lipid). The hydration of the mixed films gave dispersions with diverse liposome dimensions. As in the case of the physical properties listed above, liposome size and polydispersity failed to be described by a simple structure-function relationship.

Lipoplexes were subsequently formed by combining the positively charged liposomes with negatively charged DNA, mediated by electrostatic interactions and hydrophobic effects, in defined amine :phosphate (N/P) or molar charge ratios (+/-) of 0.5 :1, 1.5:1, 3.0:1, 5.0:1 and 10:1. DLS measurements indicated formation of lipoplexes with rather irregular hydrodynamic diameters.

Table 3: Particle sizes and polydispersity index (PDI) (DLS, 25°, detection angle 90°) of liposomes in water (A) and lipoplexes (B) in OPTI-MEM^® buffer at (+/-) molar charge ratios 0.5, 1.5, 3, 5 and 10.

A

Z-Ave, i¾

Liposomes PDI

(mn)

EPC/DOPE 580 0.5

C30-2/DOPE 215 0.3

C30-2/ Protamine/DOPE 292 0.4

B

Lipoplexes (N/P charge Z-Ave, dn

PDI

ratio) (mn)

EPC/DOPE (0.5) 1394 0.9

EPC/DOPE (1.5) 3259 0.4

EPC/DOPE (3) 1264 0.2

EPC/DOPE (5) 820 0.2

EPC/DOPE (10) 570 0.3

C30-2/DOPE (0.5) 670 0.2

C30-2/DOPE (1.5) 1206 0.9

C30-2/DOPE (3) 2889 0.9 C30-2/DOPE (5) 3633 0.7

C30-2/DOPE (10) 1329 0.6

C30-2/Protamine/DOPE

1608 0.8

(0.5)

C30-2/Protamine/DOPE

2042 0.8

(1.5)

C30-2/Protamine/DOPE (3) 3751 0.6

C30-2/Protamine/DOPE (5) 2019 0.9

C30-2/Protamine/DOPE (10) 1020 1.0

For C20-lipoplexes, sizes were typically found to be smaller with no added co-lipid when the saturated sidechain was shorter (ie. C20-14), but as the sidechain increased in length (ie. C20-18 and C20-20), formulations with cholesterol as co-lipid tended to produce smaller diameter lipoplexes. In the case of C30-lipoplexes, sizes were typically found to be smaller with no added co- lipid and largest with cholesterol as co-lipid. These large lipoplex particles are believed to be a result of aggregation and fusion of the liposomes during lipoplex formation in the buffer solution [Ref: Kedika, B.; Patri, S. V., Design, synthesis, and in vitro transfection biology of novel tocopherol based monocationic lipids: a structure- activity investigation. /. Med. Chem. 2011, 54 (2), 548-561]. Furthermore, it has been reported that DNA contributes to the electrostatic adhesion between neighboring liposomes during lipoplex formulation [Ref: Balbino, T. A.; Gasperini, A. A.; Oliveira, C. L.; Azzoni, A. R.; Cavalcanti, L. P.; de La Torre, L. G., Correlation of the physicochemical and structural properties of DNA/cationic liposome complexes with their in vitro transfection. Langmuir 2012, 28 (31), 11535-11545]. Finally, Ross et al. have reported that large lipoplexes give rise to greater cellular association and uptake in in vitro assays, compared to smaller lipoplexes, and further have reported an increase of endocytosis with increasing particle size up to 2.2 μιη and beyond for CHO-K1 cells [Ref: Ross, P. C; Hui, S. W. ; Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Therapy 1999, 6, 651-659].

A gel retardation assay was employed to study the binding interaction between C20-n cationic liposomes and DNA, as well as between C30-n liposomes and DNA. This assay revealed that, in general, DNA binding improved with increasing molar ratio of cationic lipid in the lipid/DNA lipoplex formulations; typically, near complete retention was achieved at N/P (+/-) molar charge ratio of approximately 5.0:1 or 10:1 (Fig. 8).

A DNase I degradation assay was used to determine the accessibility of the lipid-associated DNA toward nucleases. All lipid/DNA lipoplex formulations offered some degree of protection to the DNA from nuclease degradation at all charge ratios studied. Generally, longer sidechain associated with the C20-series revealed greater DNA protection, which correlates with the better DNA binding observed with C20-18 and C20-20 as indicated in the gel retardation assays. For the C30-series, when lipoplexes were formulated without a co-lipid, C30-12 and C30-20 appeared most protective. Conversely, with DOPE as co-lipid all of the C30-lipid formulations appeared equally protective of the DNA plasmid. Finally, with cholesterol as co-lipid, again C30-12 and C30-20 appeared most protective.

Each lipoplex formulation was evaluated for gene expression (Fig. 9) and cytotoxicity (Fig. 10). It is important to note that gene expression was evaluated in terms of the level of transgene expression per milligram of protein in the cell lysate, so that it was possible to obtain relatively high transfection level, even where toxicity was also high. As in the case of other chemical and physical methods of gene delivery, higher levels of transgene expression were generally accompanied by higher levels of toxicity for carotenoid formulations as well as EPC. The synthetic carotenoid lipids generally underperformed in gene expression relative to the control vector, EPC (Fig. 9). Within the C30-lipid family, the best transfection efficiencies were typically observed at intermediate molar charge ratios of 1.5 and 3. C30-20 was an exception to this rule with higher gene expression levels found with N/P ratios of 3 and 5 (or with either co- lipid). Of interest, Figure 9 reveals that this same lipid, C30-20, consistently showed the greatest overall β-gal expression in the C30-lipid family. Lipoplexes composed of the C20-lipids also revealed their greatest transgene activity without a co-lipid, or when formulated with cholesterol as co-lipid. However, unlike the C30-series, there appears to be no obvious trend in performance for the C20-series with respect to the length of the saturated sidechain. Finally, although EPC is commonly used as a formulation with the co-lipid, DOPE, overall gene expression levels were higher in the absence of a co-lipid, although the overall relationship of higher gene expression at higher N/P ratios was similar for EPC in all three formulations.

Lipoplexes were generally well tolerated by CHO-K1 cells when exposed to lipoplexes at low charge ratios (Figure 10). Furthermore, lipoplex formulations tested generally revealed a decrease in cell viability with increasing N/P (+/-) molar charge ratio. Again, formulations that incorporated C30-20 stood out from the collection of lipids that were evaluated revealing lower overall toxicity at higher N/P ratios in those formulations with an added co-lipid.

As a preliminary investigation into the morphological structures, SAXS analyses were performed on C30-20 and C20-20 lipid/DNA lipoplex formulations without colipid (Fig. 11 ; Table 4, below), and with co-lipids DOPE and cholesterol at N/P (+/-) molar charge ratio 1.5:1. These experiments revealed that the lipid-DNA packing for both the C30-20 and C20-20 lipid/DNA complex without colipid were inconclusive with respect to lamellar versus hexagonal packing, whereas those containing the co-lipid DOPE showed lamellar packing tendencies, and the formulations with the co-lipid, cholesterol most clearly presented lamellar packing. When comparing membrane structure to gene expression and cytotoxicity levels presented in Figures 9 and 10, it appears that the presence of lamellar packing correlated with cytotoxicity and was inversely correlated with gene delivery by the C20-20, but that no such relationships were observed with C30-20.

Table 4. Summary of SAXS particle packing for C20-20 and C30-20 lipid/DNA liposome and lipoplex formulations (without colipid) at (+/-) molar charge ratio 1.5:1. δ refers to the actual packing in each case, with an estimated standard deviation of typically 1 A. liposome lipoplex

lipid

packing packing distance (A)

C20-20 unimodal* inconclusive δ = -58

C30-20 unimodal inconclusive δ = -59

* Unimodal suggests no multilamellar ordering

Interestingly, at N/P (+/-) molar charge ratio 1.5 for C20-20 and C30-20 lipoplexes without co-lipid, with DOPE then cholesterol as co-lipid, we see from the SAXS data that the curve progresses from ill-defined (inconclusive) to tending towards lamellar and finally to well-defined lamellar when cholesterol is co-lipid. Subsequently, transfection experiments were performed with C20-20 and C30-20 lipoplexes formulated separately with DOPE and cholesterol as co- lipid. The transgene efficiency clearly decreased (from high to low) for vector C20-20 as the lipoplex morphology tended towards lamellar packing. There was, however, no observed correlation between lipoplex packing and transfection for the lipid C30-20 at the N/P charge ratio analyzed.

The new class of cationic glycol phospholipids, compounds of formula (III), has been synthesized with chromophoric, rigid polyene chains and flexible alkyl chains. Essential property data such as surface tension γ, aggregate concentration CM and the molecular area a_m could not be interconnected with the structure of the amphiphilic C20-n and C30-n compounds. When the homologous C20-n and C30-n amphiphiles came in contact with water, a heterologous behavior was observed caused by unpredictable self-assembling of the molecules to liposomes. Neither liposome nor lipoplex sizes were defined by trends in the chain lengths. Within the series of the highly unsaturated cationic glycol phospholipid nucleic acid carriers, the combination of the C30:9 acyl chain (i.e., a C-30 carotenoid possessing 9 double bonds) with the C20:0 (i.e., a C₂₀ alkyl group with 0 double bonds) alkyl chain showed the best in vitro DNA transfection. Elevated DNA transfer correlated with increasing saturated alkyl chain length when no co-lipid was used in the lipoplex formulations. However, this distinct structure-function relationship have not been linked to any other investigated parameters. Formulations employing the co-lipids DOPE or cholesterol revealed no apparent trend with respect to length of the saturated side chain. Besides the heterologous structural behavior of the lipid family, the multiple steps in DNA delivery may further obscure trends with respect to individual steps in the process (such as membrane fusion or endosomal escape).

Duchenne muscular dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced.

Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2- dimyristoyl-sra-glycero-3-ethylphosphocholine (EPC).

We further disclose herein the first use, to our knowledge, of carotenoid- derived cationic lipids as delivery vectors of genetic material. Two novel cationic lipids, C30-20 and C20-20 synthesized from carotenoic and retinoic acid (C30- and C20-acid), respectively, combined with a flexible icosan-l-ol chain coupled through a phosphocholine headgroup, were used to formulate lipoplexes with phosphorodiamidate morpholino (PMO) AOs designed to specifically target exon 45 skipping of the DMD mRNA and delivered into normal human skeletal muscle primary cells (hSkMCs). Skipping of exon 45 would have the potential to treat 8.1 % of DMD patients, the second highest percentage of patients after skipping of exon 51 (13%).

Accordingly, the invention can provide a method of treatment of a medical disorder in a patient wherein administration of a liposome containing a polynucleotide is medically indicated, comprising administering to the patient an effective amount of the liposome of the invention; e.g., wherein the medical disorder is Duchenne muscular dystrophy.

The carotenoid lipids C30-20 and C20-20 were synthesized from commercial C30-carotenoidester and C20-acid (retinoic acid) as described herein. All intermediates and final products were purified after each step and fully characterized by thin- layer chromatography, ultraviolet- visible

spectroscopy, low and high resolution mass spectrometry, ¹H, ¹³C and ³¹P NMR. All analytical data were found to be in accordance with known related compounds previously synthesized in our lab, and the detailed synthesis of C30- 20 and C20-20 are disclosed herein, together with the synthesis of other lipids of the C30-n and C20-n series. The structures of C30-20, C30-30, 1,2-dimyristoyl- sra-glycero-3-ethylphosphocholine (EPC) and cholesterol are shown in Figure 12.

Verification that the leash had annealed to the PMO was confirmed by agarose gel electrophoresis. Aliquots of leash alone and leashed PMO were run on a 3% agarose gel. The PMO and leash had annealed successfully as an increase in size (i.e. slower moving band) was evident in leashed PMO relative to PMO alone.

Complexation of negatively charged leashed PMO- AO with the cationic lipids results in a neutral particle once the negative charges of the nucleotides are paired with an equimolar amount of positively charged lipid molecules. Full gel retardation would then be expected once this equimolar pairing of (+/-) charges is achieved. However, structural and physicochemical differences between lipids may result in different packing properties of the various lipid/PMO-AO complexes. Complete gel retardation is not always observed with a 1 : 1 +/- charge ratio, suggesting that the properties of the various lipid/PMO AO lipoplexes are sensitive to the nature of the cationic lipid.

Lipid/PMO AO lipoplexes were prepared at various (+/-) molar charge ratios (nitrogen/phosphorus, or N/P ratios) ranging from 20:1 - 0.1 :1 for lipoplexes EPC/Chol PMO, C30-20/Chol PMO and C20-20/Chol PMO. The results of the gel retardation assay revealed that the C20-20/Chol PMO complex resulted in the highest level of retention at a charge ratio of 20:1 (Figure 13). Neither the EPC/Chol PMO or C30-20/Chol/PMO lipoplexes revealed complete retention even at charge ratios as high as 20:1.

At low (+/-) molar charge ratios (0.0 5:1 up to 0.25:1), the carotenoid lipid/PMO lipoplexes were well tolerated by hSkMCs upon visual inspection at 24 h, but some cell toxicity was evident at the higher (+/-) molar charge ratio of 0.5:1 for both carotenoid lipid/PMO lipoplexes tested (Figure 14).

To verify the efficiency of the lipoplex formulations for delivery of the targeted AO, hSkMCs were transfected with PMO AO oligomers specifically targeted for skipping exon 45 of the mRNA, and RNA was extracted after 24 h. As the levels of skipped transcript are generally very small relative to the full- length transcript, nested reverse transcriptase-PCR (RT-PCR) on the harvested RNA was required. RT-PCR was performed on 200 ng RNA from hSkMCs treated with three different lipoplex formulas, namely EPC/Chol PMO, C30- 20/Chol/PMO and C20-20/Chol/PMO. Agarose gel electrophoresis separation of products for charge ratios up to 0.5:1 for each lipid is shown in Figure 15. To normalize for RNA amount and quality within the RT-PCR assay, amplification of the housekeeping gene, ribosomal 18s was also performed (Figure 16). Equal amounts of RNA (100 ng) were subjected to RT-PCR amplification; the poor quality of RNA harvested from cells transfected with C20-20/Chol/PMO at a charge ratio of 0.5:1 is evident. This explains the failure of exon 44-48 amplification by nested RT-PCR in these samples. The poor RNA quality is likely to be the result of the toxicity seen for C20-20/Chol/PMO at the charge ratio of 0.5:1 (Figure 3, D). Semi-quantification of levels of skipping was assessed using densitometry and is shown in Figure 17. A dose-response was observed for the two carotenoid lipids, whereby the greatest exon 45 skipping for lipid C30-20 was observed at 68.3 + 25.9% at a (+/-) charge ratio of 0.5:1, and for lipid C20-20 was 29.7 + 2.3%, at a (+/-) charge ratio of 0.25:1 with 250 nM leashed PMO (Figure 17). Charge ratios beyond 1:1 for both carotenoid formulations resulted in significant cell death. At the (+/-) charge ratios tested, the two carotenoid lipids achieved greater exon 45 skipping in hSkMCs relative to the commercial lipid, EPC.

The novel cationic carotenoid lipids C30-20 and C20-20 were formulated into liposomes with the neutral co-lipid, cholesterol, as was the commercial cationic lipid, EPC. Each of these was subsequently formulated into lipoplexes containing leashed PMO capable of producing exon 45 skipping in hSkMCs; 29.7% exon skipping was achieved with C20-20/Cholesterol at a N/P (+/-) molar ratio of 0.25:1, and 68.3% with C30-20/Cholesterol, each at a N/P (+/-) molar charge ratio of 0.5:1. The C30 carotenoids performed better at lower charge ratios as compared to the commercial cationic lipid, EPC. Also, an obvious exon 45 skipping in hSkMCs 'dose-response' was observed for the C30- 20 and C20-20 cationic carotenoid lipids. This study shows that carotenoid lipids have potential as delivery vectors for antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy.

In order for the lipid-nucleic acid complex (or lipoplex) to cross the cell membrane, the complex should be charge- neutral or have an excess positive charged overall. The use of cationic lipids facilitates lipoplex formation by developing a charge-neutral complex with the negatively charged nucleic acid (DNA or siRNA). In the case of siRNA delivery, the lipoplex must escape the endosome and traffic the cytoplasm where the siRNA is taken up by the RNA- induced silencing complex (RISC), leading ultimately to the catalytic destruction of a complimentary endogenous messenger RNA (mRNA), as illustrated in Figure 18. This results in preventing the native mRNA from producing a protein product; this process is referred to as "knockdown". However, knockdown is not without restrictions when it comes to practical applications. The clinical application of RNAi is restrained by lack of tissue specificity, degradation of the complex by cellular components, and toxicity associated with the cationic lipid carrier.

Current commercially available cationic glycerolipids used for siRNA delivery are not effective enough as siRNA delivery vectors. These lipids are characterized by a common structural motif that includes a hydrophilic headgroup, linker bond, backbone (typically glycerol) and two hydrophobic tails, mainly as saturated fatty acid chains. We believe that structural modifications to the headgroup and the hydrophobic core of lipid vectors are key conditions to enhancing siRNA delivery.

Rigid lipids have been shown to self-assemble into tightly packed vesicles [21]. Such findings have lead us to the following two hypotheses: first, that the packing or self-assembling characteristics of our novel, single-chain

(i.e., of formula (IV), not comprising a phosphate group having a saturated alkyl R⁴ ester chain) rigid cationic lipids of formula (IV), 1-5 (Schemes 2 and 3) should be more related to the self-assembling characteristics of other rigid lipids, such as DC-Choi, and less like non-rigid lipids, for example EPC. Secondly, that the lipid-siRNA lipoplexes generated from our rigid carotenoid lipids of formula (IV) and those generated with the rigid control lipid, DC-Chol, would ultimately give rise to a similar therapeutic siRNA performance, but dissimilar to the non- rigid vector, EPC; hence the choice of our two positive control lipids.

We have reduced the complexity of glycero lipids by synthesizing lipid- like compounds (lipidoids) in which the non-rigid, saturated fatty chain is replaced by a rigid, polyunsaturated fatty acid directly esterified with aminoethanol derivatives.

The efficiency of siRNA delivery by the single-chain carotenoid lipid series of formula (IV) was compared with that of known cationic lipid vectors, 3 -[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1 ,2- dimyristoyl-sra-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, l,2-dioleolyl-sra-glycerol-3- phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying

(+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, cartoenoid lipids of formula (IV), specifically compounds 1, 2, 3 and 5, shown in Schemes 2 and 3, below, displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid of formula (IV) represents a new class of transfection vectors with excellent cell tolerance accompanied with encouraging siRNA delivery efficiency.

Scheme 2: Synthesis of single-chain cationic carotenoid lipids, 1-3.

30 Scheme 3: Synthesis of single-chain cationic carotenoid lipids, 4 and 5.

O

Car^OH 7

O OH

Car^O^^^{N /} "OH

4 5

Our work was inspired by the hypothesis that single-chain, rigid cationic lipids would tightly self-assemble around negatively charged nucleic acids, resulting in stable lipoplexes that perform knockdown as effectively, or better than the known rigid cationic vector, DC-Chol. Liposome particle sizing was the initial study to assess the self- assembling characteristics of our novel, single- chain rigid cationic lipids of formula (IV), compounds 1-5 of Schemes 2 and 3, with the known rigid vector, DC-Chol, and non-rigid lipid EPC.

In a first step, cationic liposomes were prepared through the sonication of a hydrated thin film of lipids formed upon elimination of ethanol by rotary evaporation. The liposome particle size data from dynamic light scattering (Figure 19) reveals a range in average liposome diameter between 100-400 nm for the majority of the lipids analyzed, with the exception of lipid 2, which resulted in average liposome diameters of 757 nm. Interestingly, liposomes composed of EPC were smaller than all of the rigid cationic lipids in this study, including DC-Chol.

The lipid headgroup choices were based on groups common to those that are presented in the literature. The positive charge associated with headgroups composed of quaternary ammonium salts is isolated mainly on nitrogen, whereas this charge can be delocalized to the N-H bond in lower order salts. This derealization of charge permits the surrounding water molecules to reduce the positive charge through hydrogen bonding interactions. This interaction of HBr salts (1 and 4 based liposomes) with water participates in the stabilization of liposomes in the aqueous media. Liposomes prepared from lipids 3-5 containing hydroxyl moieties at the headgroups may exhibit a similar stabilizing effect through the interaction with surrounding water molecules. In contrast, lipid 2 containing a quaternary ammonium cannot participate in such stabilization, and thus gave rise to the formation of aggregates upon hydration.

Combining the negatively charged siRNA with the cationic liposomes resulted in lipoplex formation initially mediated by electrostatic interactions and subsequently by hydrophobic effects. The carotenoid lipid/siRNA lipoplexes assemble into nanosized particles ranging from 100-550 nm diameters, where the smallest particles correspond to lipoplexes with a (+/-) molar charge ratio of 2.5, and largest particles at a (+/-) molar charge ratio of 10:1. Aggregates with size ranging from 1 to 5 μιη were detected in all lipoplex samples, particularly with lipoplexes prepared from the carotenoid lipid 2. Only the major populations formed by the submicron size particles were taken into consideration in the calculation of lipoplex size.

The efficiency of the lipoplexes (the ability of carried GL2 anti-luciferase to knockdown the luciferase expression compared to that of non treated cells) was investigated by a luciferase knockdown assay in HR5-CL11 cells, stably transfected with the luciferase reporter via a tetracycline controlled

transcriptional trans- activator. Cells were transfected with lipoplexes for 4 h before the media was replaced with complete growth media containing antibiotics followed by incubation at 37 °C, 5% C0₂ for 48 h before realizing the assay.

In general, the data revealed variable knockdown performance for the different vectors assayed (Figure 20). Lipoplexes composed from lipids 4 did not result in knockdown activity, whereas those composed of carotenoid lipids 1, 2, 3 and 5 revealed significant anti-luciferase capacity for the GL2 anti-luciferase treated group at one or more (+/-) charge ratios studied. Lipoplexes composed from lipid 2 revealed a moderate anti-luciferase capacity at (+/-) charge ratio 5 for the GL2 anti-luciferase treated group (p < 0.05). Formulations that displayed significant efficiency towards luciferase knockdown in GL2 treated HR5 CL11 cells over no n- treated cells p < 0.01) are those containing lipid 1 at the lowest (+/-) charge ratio studied, lipid 2 with (+/-) charge ratios of 7.5, lipid 5 at the highest (+/-) charge ratio studied. All lipoplexes containing lipid 3 formulated with (+/-) charge ratios of

2.5-10 displayed significant efficiency towards luciferase knockdown in GL2 treated HR5 CLl 1 cells. Cells treated with control siRNA-containing formulations revealed equal or greater luciferase signal than that of cells treated with GL2.

Although these lipids revealed promising efficiencies within particular (+/-) charge ratios, none proved as efficient in knocking down luciferase expression as the control lipids, DC-Chol and EPC, two known non- viral gene delivery agents.

It is well documented that the charge ratio of the positive lipid to negative nucleic acid cargo affects toxicity, with higher (+/-) charge ratios generally being more toxic. The toxicity of lipoplexes may cause inflammatory reactions by interaction with the reticuloendothelial system (RES). The cytotoxicity associated with the carotenoid-based lipoplexes and those of DC- Chol and EPC with (+/-) molar charge ratios ranging from 2.5 to 10, was determined on the HR5-CL11 cell line after a 48 h incubation at 37 °C with 5% C0₂ using the MTS assay. Results reported in Figure 21 reveal concentration- dependent cytotoxicity associated with all lipoplex formulations, with the exception of the carotenoid lipid 4, and EPC.

Lipoplexes containing lipid 2, as well as those formulated from 3-5 (functionalized with hydroxyl moieties at the lipid headgroup) were found to be cytotoxic beyond (+/-) molar charge ratio 5, with less than 50% cell viability. Lipoplexes containing lipid 1 and EPC were very well tolerated by the HR5 CLl 1 cells at all charge ratios studied. Those containing DC-Chol exhibited high cytotoxicity beyond charge ratio 2.5.

Herein, five novel lipids were synthesized having a common rigid C30- carotenoid hydrophobic domain, while differing in the nature of the amphiphilic headgroups, and were evaluated for their ability to delivery siRNA.

Lipids 1 and 2, with tertiary and quaternary amine headgroups, respectively, revealed knockdown efficiencies at various charge ratios. Lipid 1 displayed significant knockdown only at (+/-) charge ratio 2.5 whereas Lipid 2 produced significant knockdown at (+/-) charge ratios 5 and 7.5. Lipid 1 was significantly less toxic than 2 for all (+/-) charge ratios studied.

Cationic lipids containing amine headgroups functionalized with an hydroxyethyl moiety have been shown to enhance transfection efficiency, hence the justification for lipids 3-5. This might explain the significant knockdown efficiency for lipid 3 at all (+/-) charge ratios, not seen with lipids 1 and 2. Adding a second hydroxyethyl group (as is the case for lipids 4 and 5) did not further enhance the knockdown efficiency, but rather increased the cytotoxicity.

One limitation of this study to note is that two of the five cationic carotenoid lipids, namely 3 and 5, support an iodide counter ion while the remaining three carotenoid lipids have a bromide counter ion. This could potentially complicate the interpretation of results when comparing the relative knockdown efficiencies.

The luciferase expression (relative light units, RLU) was normalized by the total protein content (absorbance at 562nm, A562) to decouple cytotoxicity from luciferase knockdown.

DC-Choi appeared to exhibit a superior knockdown efficiency over all charge ratios studied, however, it was revealed through the MTS assay that DC- Chol exhibited high cytotoxicity beyond charge ratio 2.5, and therefore the knockdown results may be taken with precaution as may be more a function of cell death rather than RNA interference.

Lipids 1, 2, 3 and 5 combine good cell tolerance (particularly lipid 1) with knockdown activity and therefore represent suitable candidates for further investigation.

The literature clearly indicates that cationic lipid gene transfer vectors with two hydrophobic chains are generally more active than those with a single single-chain. Our preliminary findings within this study suggest that the novel, single-chain rigid cationic carotenoid lipids are able to complex and deliver siRNA across the cell membrane of eukaryote cells. However, the results from this preliminary study comparing our single-chain rigid lipids with the rigid control lipid DC-Choi do not support our hypotheses of an influence on liposome packing and siRNA delivery efficiencies.

Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed good knockdown efficiency with HR5-CL11 cells at defined (+/-) molar charge ratios. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied; exceeding the cell viability of both control lipids, DC-Choi and EPC. These novel, single-chain rigid carotenoid-based cationic lipids represent a new class of transfection vectors with good cell tolerance accompanied with encouraging in vitro lucif erase knockdown activity in HR5-CL11 cells. Our efforts remain ongoing towards the enhanced efficiency of these single-chain transfection vectors through modification at the lipid headgroup, counter ion and lipoplex formulation.

Compounds of formula (V) can be prepared according to Scheme 4, in conjunction with ordinary knowledge and skill in the art. Cationic phospholipids have been prepared by esterification of P-apo-8'-carotenoic acid (1) or retinoic acid (2) with ( ?)-glycerophosphocholine (lysoPC:n) with variable alkanoic chain lengths (n = 14, 16, 18) in the presence of dicyclohexadecylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). Alkylation of the resulting zwitterionic compounds with ethyltriflate gave C20Glyc-n, n = 14, 16, 18 and C30Glyc-14. Variations in groups R¹, R², R³, and R⁵, can be effected by selection of the appropriate acyl Lyso PC starting material, and the phosphate ester group R⁴ can be varied by selection of the appropriate triflate or other alkylating agent.

Scheme 4: Synthesis of cationic (7?)-glycerophospholipids with variable polyenoic and alcanoic chain lengths.

Examples

Synthesis

NMR spectra (1H and ¹³C) were acquired on a Bruker Avance DPX 400

MHz and Bruker Avance 600 MHz with CDC1₃ unless otherwise stated. UV-VIS spectra were recorded in CH2CI2 using a Single Beam Thermo Spectronic, Helios. Mass spectra data were acquired on a MAT 95XL, TermoQuest Finnigan mass spectrometer equipped with an electron ionization (EI) or electrospray ionization (ESI) resource. Flash column chromatography (flash-CC) was performed with silica gel (Woelm Pharma 60 mesh) or neutral alumina (II-III Brochmann activity, EcoChrom, 100-150 mesh). Surface tension was determined using a Wilhelmy (Pt) plate on a Kriiss Tensiometer K100. Particle size was measured by dynamic light scattering instruments (NTNU: ALV DLS/SLS-5022F compact goniometer with ALV-5000/E multiple digital correlator, ALV Langen, Germany. WCMCQ: Zetasizer APS, Malvern

Instruments, Worcestershire, UK). The aggregation concentration c_ag was found by dissolving the compounds in the indicated solvents. H₂0 was added in 100 μΕ amounts and monitored VIS-spectroscopically for aggregate formation. Inversely, the compounds were dispersed in H₂0 and organic solvent was added until disruption of the aggregates.

Lipids EPC and cholesterol were obtained from Avanti Polar Lipids. PMO AO h45A30/l (sequence available on request) was purchased from Gene Tools, Philomath, OR, USA, complementary leash h45A30/lL (sequence available on request) and RT-PCR primers from Eurofins MWG Operon (Ebersberg, Germany). Normal human skeletal muscle primary cells (hSkMCs) were purchased from TCS cellworks (Buckingham, UK), skeletal muscle cell growth and differentiation media plus supplements from PromoCell GmbH (Heidelberg, Germany), GeneScript RT-PCR system kit and 2 x PCR Master Mix with cresol red from GeneSys Ltd. (Camberley, Surrey, UK). QIAshredder kit and RNeasy Mini kit were purchased from Qiagen Ltd. (Crawley, UK). Agarose, buffers and antibiotics were purchased from Invitrogen Ltd. (Paisley, UK). All solvents and chemical reagents were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Dichloromethane was obtained from Alfa Aesar (West Hill, MA, USA).

The carotenoid components of the compounds of the invention can be synthesized as described herein starting with commercially available (e.g.,

for compounds wherein the carotenoid moiety is of formula (IIA), and starting with

99-20-5)

for compounds wherein the carotenoid moiety is of formula (IIB).

Specific synthetic procedures are provided below. Other reagents and techniques are either described in the present application or are within the knowledge of the person of ordinary skill in the art of organic synthesis.

Ethyl P-apo-8'-carotenoate (CAS 1109-11-1) was obtained from Dr. H.

Ernst, BASF SE, Ludwigshafen, Germany. The control cationic lipid 1,2- dimyristoyl-sn-glycero-3-ethylphophocholine (EPC) and co-lipid 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE) were obtained from Avanti Polar Lipids (Alabaster, USA). Protamine sulfate was purchased from Sigma Aldrich (Taufkirchen, Germany). The Chinese hamster ovary-Kl (CHO-K1) cell line was purchased from Health Protection Agency Culture Collections (Salisbury, UK). Cell culture media, antibiotics and Lipofectamine 2000 were purchased from Invitrogen Ltd. (Paisley, UK). Plasmid DNA containing the β- galactosidase gene, pCMV iacZnlsl2co was obtained from Marker Gene Technologies, Inc. (Oregon, USA). Beta-Glo® Assay System, CellTiter 96®AQueous One Solution Cell Proliferation Assay and Passive lysis buffer were purchased from Promega (Madison, WI, USA), BCA Protein Quantitation Assay was purchased from Pierce Biotechnology (Thermo Fisher Scientific, Rockfort, IL, USA). Dichloro methane was obtained from Alfa Aesar (West Hill, MA, USA). Unless otherwise stated, all solvents and chemical reagents were obtained from Sigma Aldrich (St. Louis, MO, USA).

Surface tension measurements was obtained using a Wilhelmy (Pt) plate on a Kriiss Tensiometer K100. The tensiometric curves were interpreted by regression analysis in order to determine the critical aggregation concentration

CM. With CM, yc„ > surface pressure π, surface concentration Γ, area per molecule at the filled monolayer a_m, the free energy of aggregation AG and of adsorption AG°d , the surfactant performance indicator AMER(Skrylev et al. , 2000) and the equilibrium constants for aggregation and absorption k_ag and k_ad were calculated (Foss et al., 2005b; Foss et al., 2005c). Γ and a_m were assessed assuming that the molecules do not dissociate in water (one species) or that they completely dissociate (two species). The values for a_m supposing complete dissociation were too high and in variance with molecular calculations.

Aggregate size (hydrodynamic radius ¾). The aggregate size of aqueous C30-2 dispersions at two different concentrations (0.004 mg/mL and 0.06 mg/mL) were analyzed by Dynamic Light Scattering (DLS) instrument ALV DLS/SLS-5022F compact goniometer with ALV-5000/E multiple τ-digital correlator, ALV Langen, Germany, at three different scattering angles (Santos and Castanho, 1996).

Stock solutions of novel cationic lipid C30-2 and commercial cationic lipid EPC and co-lipid DOPE were made by dissolving a known amount of each lipid in CH2CI2 in a round-bottom flask. The solutions were placed on a rotary evaporator for 1 h to obtain a film. The film was dissolved in a known amount of anhydrous EtOH in order to achieve a 1 mM stock, and subsequently stored at -80 °C. A 10 mg/ml stock of protamine sulfate in sterile water was made and subsequently used in liposome preparations. A 3:2 molar ratio of cationic lipid (either carotenoid C30-2 or control lipid EPC) to co-lipid, DOPE, in ethanolic solutions were prepared separately and evaporated under reduced pressure to generate thin films. The lipid films were hydrated with a known amount of sterile water to give 2 mM final hydrated stock solutions, which were stored overnight at 4 °C. Before use, the hydrated stocks were warmed to 37 °C and sonicated for 30 minutes. In the case of the C30-2/protamine/DOPE liposome, protamine sulfate solution (500 μΕ; 0.005gm; 0.98xl0^~6 mol) was added directly onto the C30-2/DOPE thin film (prepared as above) and stored overnight at 4 °C. Before use, the hydrated stock was warmed to 37 °C and sonicated for 30 minutes.

Lipoplexes of concentrations 0.081 mM, 0.243 mM, 0.486 mM, 0.81 mM and 1.62 mM, corresponding to the N/P (+/-) molar charge ratios of 0.5:1, 1.5:1, 3:1, 5.0:1 and 10.0:1, respectively, were prepared from a 2 mM C30- 2/DOPE and EPC/DOPE liposome stocks. OPTI-MEM buffer (57.6 μΕ) and pDNA in Elution solution, pH 8, (14.4 μΕ; 250 ng/μΕ) were first combined, followed by the addition of an equal volume of corresponding liposome (72 μΕ) to this and mixed. These lipoplex formulations were incubated at Room Temperature (RT) for 30 min. Forty-eight microliters of lipoplex formulation was used for the gel assays and to each of the remaining lipoplex formulations, 204 μΕ of OPTI-MEM was added and subsequently used for transfections. From the C30- 2/protamine/DOPE liposome, lipid/pDNA complexes were generated corresponding to the N/P (+/-) molar charge ratios of 0.5:1, 1.5:1, 3:1, 5.0:1 and 10.0:1, based solely on the positive charge of C30-2 cationic lipid. OPTI-MEM buffer (57.6 μΕ) and ρΌΝΑ in Elution solution, pH 8, (14.4 μΕ; 250 ng/μΕ) were first combined, followed by the addition of an equal volume of liposome (72 μΕ) and mixed. These lipoplex formulations were incubated at RT for 30 min. Forty- eight microliters of lipoplex formulation was used for the gel assays and to each of the remaining lipoplex formulations, 204 μΕ of OPTI-MEM was added and subsequently used for transfections. The hydrodynamic diameter, i¾_, of liposomes and lipoplexes was measured by quasi-elastic light scattering with a Zetasizer APS (Malvern Instruments, Worcestershire, UK) at 25 °C with a detection angle of 90°. All data are the mean + standard deviation (SD) of three measurements.

To 20 μL· of the lipoplexes, 2 μL· of the gel loading dye (6X) was added and mixed by pipetting. Eighteen microliters of each sample was then loaded onto a 1% agarose gel impregnated with ethidium bromide and run at 105 V for 1 h in lx TBE buffer. The migration of pDNA complexed with the cationic lipids was impeded in the electric field. The pDNA bands were observed using a Geliance transilluminator.

Twenty microliters of the lipoplexes was incubated with DNase 1 (1 μί) at 37 °C for 1 h. After incubation, 5% SDS (4 μΚ) was added and incubated for 30 min, followed by 2 of gel loading dye (6x). Eighteen microliters of each sample was then loaded onto a 1 % agarose gel impregnated with ethidium bromide and run at 105 V for 1 h in lx TBE buffer. The pDNA bands were observed using a Geliance transilluminator.

CHO-K1 cells were grown in RPMI media supplemented with 10% fetal calf serum and 100 U/mL of penicillin/streptomycin and 0.25 μg/ml amphotericin B. Cells were seeded 48 h before transfection onto opaque and transparent 96-well plate at a density of 10⁴ cells per well and incubated at 37 °C in presence of 5% C0₂ atmosphere. Cells were grown to 80% confluence before being washed with lx PBS and incubated with 45 μΐ of each lipid-pDNA complex in triplicate for 4 h at 37 °C in the presence of 5% C0₂ atmosphere. Complexes were then removed and the cells washed with lx PBS before adding 100 μΐ of RPMI media. Cells were left to incubate for an additional 44 h.

Following the incubation, transfection and cytotoxicity assays were performed according to the below mentioned protocols.

Forty-eight hours after the application of lipoplexes, β-galactosidase activity was determined using a Beta-Glo^® Assay System (Promega), treated cells in the opaque 96-well plate were washed with lx PBS, then 50 μΕ of

DMEM (phenol red-free media) was added to each well followed by 50 μΕ of

Beta Glo™ working solution, prepared according to the manufacturer's directions (Promega) was added to each well and mixed by pipetting. After 1 h incubation at RT, luminescence was then read on a Victor Envision high throughput plate reader. β-Galactosidase activity was expressed as relative light units produced by the luminescence of luciferin, which was normalized for protein content.

Total protein content was measured using Pierce^® BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Forty-eight hours after the application of lipoplexes, treated cells in the transparent 96-well plate were washed with lx PBS, 10 μL· of passive lysis buffer (Promega) was added to each well. Plates were wrapped with plastic wrap and incubated at RT for 30 min. BCA working reagent (200 μί), prepared according to the manufacturer's directions, was then added to each well, gently mixed by pipetting, and incubated at RT for 1 h prior to reading at 562 nm on a Victor Envision plate reader. A calibration curve obtained from a bovine serum albumin standard solution was used to determine cellular protein content per well.

The cytotoxicity associated with the lipoplex formulations at N:P (+/-) molar charge ratios ranging from 0.5:1 to 10:1 was evaluated using the MTS (3- (4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) assay. Forty-eight hours after the application of lipoplexes, CHO- Kl treated cells in the transparent 96-well plates were washed with lx PBS, 50 μΐ. of DMEM (phenol red-free media) followed by 10 μΐ. of CellTiter96^® Aqueous One Solution Cell Proliferation Assay (Promega) was added to each well and mixed by gentle rocking. The plates were incubated further for 1 h at 37 °C. The absorbance of converted dye, which correlates with the number of viable cells, was measured at 490 nm using a Victor Envision high throughput plate reader. The percentage of viable cells was calculated as the absorbance ratio of treated to untreated cells.

In order to evaluate the pDNA transfection efficiency of lipoplexes formulated with the cationic lipids, cells were transfected with lipoplexes for 4 h, then replaced with complete growth media, followed by incubation for a further 44 h at 37 °C in the presence of 5% C0₂ atmosphere before assaying β- galactosidase expression using the Beta-Glo^® Assay System (Promega).

Synthetic procedures

2-Hydroxy ethyl- -apo-8'-carotenoate

-Apo-8'-carotenoic acid (1.00 g, 2.31 mmol), DCC (1.5 eq, 716 mg, 3.47 mmol) and a minute amount of DMAP dissolved in dry CH2CI2 was added to dry ethylene glycol (10 mL) and the mixture stirred at room temperature under N2 for 2 days. Extraction with water (3 x 50 mL), drying over anhydrous Na2S0₄ and concentration gave a residue, which was dissolved in cold acetone (5 mL) and filtered to remove DCC-urea. Flash-CC on silica with a toluene-acetone gradient gave glycol monoester 2-Hydroxyethyl- -apo-8'-carotenoate (892 mg, 81%). TLC (toluene/acetone/ MeOH 6:1 : 1 v/v): R_f = 0.51. UV/Vis (CH₂C1₂):

= 456 nm. ^lU NMR: 4.305 (t, 2H, H-Cl), 3.827 (t, 2H, H-C2), carotenoyl part in accordance with -Apo-8'-carotenoic acid. ¹³ C NMR: 66.448 (CH₂, CI), 61.730 (CH₂, C2). HRMS: C₃2H4₄0₃ calcd. 476.32905 (M⁺), found 476.32908. 2-Bromoethyl dichlorophosphate (Hansen et al., 1982; Modro and Modro, 1992)

Freshly distilled 2-bromoethanol (1.05 eq, 6.08 g, 50 mmol) dissolved in dry CH2CI2 (5 mL) was added drop-wise during 1 h to an ice-cooled solution of freshly distilled POCl₃ (7,35 g, 48 mmol) dissolved in dry CH₂C1₂ (10 mL). The reaction mixture was refluxed for 5 h. Vacuum-distillation (b.p. 66-69 °C, 0.4 mbar) gave 2-Bromoethyl dichlorophosphate (10.4 g, 90%). H NMR: 4.550 (dt,

³JH-P = 10.43 Hz, 2H, H-Cl), 3.580 (t, 2H, H-C2). ¹³C NMR: 70.030 (b, CH₂, CI), 27.500 (b, CH₂, C2). ³¹P NMR: 7.440. MS (EI): 241 + 243 (1 : 1, M-l); HRMS : calcd. for Cz^BrClzOzP 241.8458 (M-l); found 241.84549.

2-((2-bromoethoxy)( ethoxy)phosphoryloxy) ethyl β-αρο-8 '-carotenoate

2-Bromoethyl dichlorophosphate (5 eq, 1.473, 5.10 mmol) was dissolved in dry diethyl ether (15 mL) and cooled on ice. Dry triethylamine (5 mL), followed by glycolmonoester 2-Hydroxyethyl-P-apo-8'-carotenoate (486.6 mg, 1.02 mmol) dissolved in dry diethyl ether (20 mL), was added drop- wise and the mixture was refluxed for 5 h. Dry ethanol (5 mL) was added and the reaction mixture was stirred at 20 °C overnight. Extraction with water (3 x 20 mL), drying over Na₂S04 gave a residue, which, after flash CC on silica with a toluene-acetone gradient gave 2-((2-bromoethoxy)(ethoxy)phosphoryloxy)ethyl -apo-8'- carotenoate (641 mg, 91%). TLC (toluene/acetone/MeOH 6/1/1 v/v): R_f = 0.59. UV/Vis (CH₂C1₂):

456 ran. 1H NMR: 4.290 (dt, 2H, H-Cl), 4.360 (dt, 2H, H-C2), 4.290 ppm (dt, 2H, H-Cl), 3.510 (dt, 2H, H-C2'), 4.150 (dq, 2H, H-Cl), 1.300 (dt, 3H, H-C2"), carotenoyl part in accordance with -Apo-8'-carotenoic acid. ¹³C NMR: 66.700 (d, ²J_C-P = 5.39 Hz, CH₂, CI), 63.070 (d, ³J_C-P = 7.05 Hz, CH₂, C2), 65.780 (d, ²J_C-P = 5.80 Hz, CH₂, CI), 29.440 (d, ³J_C-P = 7.46 Hz, CH₂, C2'), 64.470 (d, ²J_C-P = 5.80 Hz, CH₂, CI), 16.120 (d, ³J_C-P = 6.63 Hz, CH₃,

C2"), carotenoyl part in accordance with -Apo-8'-carotenoic acid. ³¹P NMR: - 0.385. MS: 690.2 + 692.2 (1 :1 , M⁺); HRMS: C36H₅₂Br0₆P calcd. 692.26724 (M⁺), found 692.76766.

( Choline)(ethoxy)phosphoryloxy)ethyl β-αρο-8 '-carotenoate (C30-2).

Bromo compound 2-((2-bromoethoxy)(ethoxy)phosphoryloxy)ethyl -apo-8'- carotenoate (180 mg, 0.26 mmol) was dissolved in CHCl₃/i-PrOH/DMF (3:5:5 v/v, 30 mL). NMe₃ (45% in water, 8 mL) was added ad the mixture stirred at room temperature under N₂ for 4 days. Flash-CC on neutral Al₂03 with 15% methanol gave C30-2 (167 mg, 78%). TLC (CHCl₃/MeOH/H₂0 4:5 :1 v/v): R/ = 0.15. UV/Vis (CH₂C1₂): Lax = 458 nm. ^XH NMR: 4.230 (dt, 2H, H-Cl), 4.260 (dt, 2H, H-C2), 4.440 (dt, 2H, H-Cl'), 4.060 (dt, 2H, H-C2'), 3.430 (s, 9H, H- C47H-C57H-C6'), 4.050 (dq, 2H, H-Cl "), 1.250 (dt, 3H, H-C2"), carotenoyl part in accordance with -Apo-8'-carotenoic acid. ¹³C NMR: 65.930 (d, ²Jc-p = 4.6 Hz, CH₂, CI), 62.570 (d, ³J_C-P = 6.2 Hz, CH₂, C2), 61.170 (d, ²J_C-P = 4.7 Hz, CH₂, CI'), 64.990 (d, ³J_C-_P = 6.7 Hz, CH₂, C2'), 53.990 (CH₃, C47C57C6'), 64.930 (d, ²JC-P = 6.4 Hz, CH₂, CI "), 16.150 (d, ³J_C-_P = 7.07 Hz, CH3, C2"), carotenoyl part in accordance with -Apo-8'-carotenoic acid. ³¹P NMR: -1.194. HRMS : CsgHeiNOeP calcd. 670.4237 (M⁺), found 670.4231. -bromoethyl)(hexyl)(2-hydroxyethyl)phosphate 4-6

2-Chloro-l ,3,2-dioxaphospholane (1.25 eq., 2.00 g, 15.8 mmol) was dissolved in dry CH₂CI₂ (50 mL) and cooled on ice. Dry triethylamine (1.5 eq., 1.94 g, 19.2 mmol) and 1-hexanol (1.31 g, 12.8 mmol) were introduced drop-wise and the mixture refluxed under N₂ for 16 h. After cooling to -20 °C, B¾ was added until the solution became permanent slight yellow. Dry triethylamine (2 mL) and ethyleneglycol (5 mL) were added and the mixture refluxed for 12 h. Extraction of the mixture with water (3 x 50 mL), drying with anhydrous Na₂S0₄, and vacuum concentration gave a residue, which, after flash-(2- bromoethyl)(hexyl)(2-hydroxyethyl)phosphate CC on silica with hexane/acetone 1 :1 v/v, gave (1.38 g, 32%). TLC (toluene/acetone/MeOH, 6: 1 :1 v/v): R_f = 0.42. ^XH NMR: 4.192 (dt, 2H, H-l), 3.838 (dt, 2H, H-2), 4.348 (dt, 2Η,Η-Γ), 3.556 (dt, 2H, H-2^'), 4.109 (dt, 2H, H-l ^"), 1.698 (m, 2H, H-2^"), 1.376 (m, 2H, H-3 ^"), 1.309 (m, 2H, H-4"), 1.315 (m, 2H, H- 5 "), 0.891 (t, 3H, H-6"). ¹³C NMR: 70.012 (d, ²JC-P = 5.5 Hz, CH₂,C-1), 62.100 (d, ³J_C-P = 5.5 Hz, CH₂, C-2), 66.898 (d, ²JC-P = 5.5 Hz, CH₂, C- r), 29.530 (d, ³J_C-_P = 7.7 Hz, CH₂, C-2^'), 68.664 (d, ²JC-P = 6.6 Hz, CH₂, C-l ^"), 30.167 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^"), 25.047, (CH₂, C-3 ^"), 31.273 (CH₂, C-4^"), 22.505 (CH₂, C-5 ^"), 13.977 (CH₃, C-6^"). ³¹P NMR: - 0.501. HRMS ^[-^]: CicfeBrOjP calcd. 333.0461 (M+H), found

333.0477. -brotnoethyl)(dodecyl)(2-hydroxyethyl)phosphate (4-12).

Phosphate triester 4-12 was obtained (250 mg, 5%) from 1-dodecanol (2.39g 12.8 mmol) as described for 4-6. TLC (toluene/acetone/MeOH 6:1 :1 v/v): R_f = 0.38. ^XH NMR: 4.179 (dt, 2H, H-1), 3.824 (dt, 2H, H-2), 4.331 (dt, 2H, H-1'), 3.553 (dt, 2H, H-2'), 4.096 (dt, 2H, Η- ), 1.692 (m, 2H, H-2^'), 1.347 (m, 2H, H- 3 , 1.3-1.1 (12H, H-4^'-H-9^"), 1-262 (m, 2H, H-10^"), 1-274 (m, 2H, H-l l ^"), 0.874 (t, 3H, H-12'0. ¹³C NMR: 69.857 (d, ²J_C-p = 6.6 Hz, CH₂, C-l), 61.821 (d, ³Jc-p = 5.5 Hz, CH₂, C-2'), 66.825 (d, ²J_C__P = 5.5 Hz, CH₂, C-l), 29.697 (d, ³J_C-_P = 6.6 Hz, CH₂, C-20, 68.583 (d, ²J_C-_P = 6.6 Hz, CH₂, C-l ^"), 30.137 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^"), 25.317 (CH₂, C-3 ^"), 29.6-29.0 (6C, CH₂, C-4^"-C-9^"), 31.837 (CH₂, C-10^'0, 22.614 (CH₂, C-l l ^"), 14.050 (CH₃, C-12^"). ^3IP NMR: 0.253. HRMS ^[-^]: Ci₆H₃₄Br0₅P calcd. 417.1400 (M+H), found 417.1420. -bromoethyl)(tetradecyl)(2-hydroxyethyl)phosphate (4-14).

Phosphate triester 4-14 was obtained (1.22 g, 21%) from 1-tetradecanol (2.74 g, 12.8 mmol) as described for 4-6. TLC (toluene/acetone/MeOH 6:1 :1 v/v): R_f = 0.45. ^lU NMR: 4.189 (dt, 2H, H-1), 3.833 (dt, 2H, H-2), 4.341 (dt, 2H, Η-Γ), 3.549 (dt, 2H, H-20, 4.099 (dt, 2H, Η- ), 1.697 (m, 2H, H-2^"), 1-355 (m, 2H, H-3 ^"), 1.3-1.1 (16H, H-4^"-H-l l ^"), 1.261 (m, 2H, H-12^'0, 1-261 (m, 2H, H- 13 ^"), 0.875 (t, 3H, H-14^"). ¹ C NMR: 69.923 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l), 61.990 (d, J_C-_P = 5.5 Hz, CH₂, C-2), 66.832 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l'), 29.581 (d, J_C-_P = 6.6 Hz, CH₂, C-2'), 68.612 (d, ²J_C-_P = 6.6 Hz, CH₂, C-l ^"), 30.167 (d, ³J_C-_P= 6.6 Hz, CH₂, C-2"), 25.339 (CH₂, C-3 "), 29.65-29.50 (8C, CH₂, C-4--C-11 ^"), 31.874 (CH₂, C-12^"), 22.643 (CH₂, C-13 ^"), 14.079 (CH₃, C-14"). ³¹P NMR: -0.577. HRMS ^[-^]: Ci₈H₃₈Br0₅P calcd. 445.1713 (M+H), found 445.1729. -bromoethyl)(hexadecyl)(2-hydroxyethyl)phosphate (4-16)

2-Bromoethyl dichlorophosphate (1.49 eq., 2.97 g; 0.0122 mol) was dissolved in anhydrous CH₂C1₂ (20 mL) and cooled to 0 °C. Anhydrous triethylamine (1.98 eq; 1.642 gm; 0.016 mol) was dissolved in anhydrous CH2CI2 (10 mL) and drop wise added to the solution. 1-Hexadecanol (1 eq., 2 g, 0.0082 mol) dissolved in anhydrous CH2CI2 (20 mL) was drop wise added and the resulting mixture was refluxed for 5h. Ethylene glycol (2.19 eq., 1.11 g; 0.0082 mol) was added and the mixture refluxed for 22 h, extracted with H₂0, dried over Na₂SC>4 and concentrated in vacuo. Purification by flash-CC on silica with hexane: acetone 1 :1 v/v gave 4-16 (1.51 g, 39%). TLC (toluene/acetone/MeOH, 6:1 :1 v/v): R_f = 0.47.

^XH NMR: 4.111 (dt, 2H, H-l), 3.742 (b, t, 2H, H-2), 4.300 (dt, 2H, Η- ), 3.508 (t, 2H, / = 12.2 Hz, H-2^'), 4.055 (dt, 2H), 1.697 (m, 2H), 1.355 (m, 2H), 1.3-1.1 (20H), 1.261 (m, 2H), 1.261 (m, 2H), 0.875 (t, 3H, / = 13.2 Hz). ¹³C NMR: 69.718 (CH₂, C-l), 61.525 (CH₂, C-2), 66.822 (CH₂, C-Γ), 29.507 (CH₂, C-2^'), 68.493 (CH₂), 30.176 (CH₂), 25.330 (CH₂), 29.52-29.50 (IOC, CH₂), 31.855 (CH₂), 22.615 (CH₂), 14.042 (C¾). ³¹P NMR: -0.576. HRMS: C₂₀H4₂BrO₅P calcd. 473.2024 (M+H), found 473.2024. -bromoethyl)(octadecyl)( 2-hydroxyethyl jphosphate ( 4-18)

Phosphate triester 4-18 was obtained (1.87 g, 34%) from 1-octadecanol (3.00 g, 11.0 mmol) as described for 4-16. TLC (toluene/acetone/MeOH, 6 : 1 : 1 v/v) : R_f = 0.44.

*H NMR: 4.137 (dt, 2H, H-l), 3.856 (br, t, 2H, H-2), 4.314 (dt, 2H, Η-Γ), 3.578 (t, 2H, / = 12.4 Hz, H-2'), 4.086 (dt, 2H), 1.743 (m, 2H), 1.375 (m, 2H), 1.3-1.1 (24H), 1.154 (m, 2H), 1.258 (m, 2H), 0.889 (t, 3H, / = 13.6 Hz). ¹³C NMR: 69.716 (CH₂, C-l), 61.535 (CH₂, C-2), 66.825 (CH₂, C-l , 29.512 (CH₂, C-2^'), 68.399 (CH₂), 30.133 (CH), 25.321 (CH₂), 29.52-29.51 (12C, CH₂), 31.832 (CH₂), 22.598 (CH₂), 14.012 (CH₃). ³¹P NMR: -0.512. HRMS: C₂₂H4₆Br0₅P calcd. 501.2333 (M+H), found 501.2334. (2-bromoethyl)(icosyl)(2-hydroxyethyl)phosphate (4-20) 2" 4" 6" 8" !0^'- 12^" 14" id^" 1ST ->Q" 1 ^" 3^" 5" ?- 9" ir" 13" IS" J.?^'" 19^"

Phosphate triester 4-20 was obtained (44g, 21%) from 1-icosanol (3.82g, 12.819 mmol) as described for 4-6. TLC (toluene/acetone/MeOH 6:1 :1 v/v, R_f= 0.47. NMR: 4.116 (dt, 2H, H-l), 3.760 (dt, 2H, H-2), 4,276 (dt, 2H, Η- ), 3.498 (dt, 2H, H-2 , 4.033 (dt, 2H, H-l ^"), 1.628 (m, 2H, H-2^"), 1.301 (m, 2H, H-3 ^"), 1.3-1.1 (28H, H-4^"-H-17^"), 1 - 196 (m, 2H, H-18^"), 1.219 (m, 2H, H-19^"), 0.819 (t, 3H, H-20^");¹³C NMR: 69.740 (d, ²J_C._P = 5.5 Hz, CH₂, C-l), 61.689 (d, ³JC-P = 5.5 Hz, CH₂, C-2), 66.722 (d, ²J_C-_P = 5.5 Hz, CH₂, C-Γ), 29.243 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^'), 68.444 (d, ²J_C-_P = 6.6 Hz, CH₂, C-l ^"), 30.079 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^"), 25.259 (CH₂, C-3 ^"), 29.6-29.0 (14C, CH₂, C-4^"-C-17^"), 31.793 (CH₂, C-18 "), 22.555 (CH₂, C-19"), 13.970 (CH₃, C-20"). ³¹P NMR: - 0.017. HRMS (ESI): C₂₄H₅₀BrO₅P calcd. 528.2579, found 528.260.

2-((2-bromoethoxy) (hexyloxy) phosphoryljethoxy-retinoate (10-6)

Retinoic acid (200 mg, 0.666 mmol), 4-6 (266 mg, 0.799 mol),

chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP, 1 ,25 eq., 349 mg, 0.868 mmol), N-ethyl diisopropylamine (DIEA, 0.65 eq., 58 mg, 0.451 mmol), and DMAP (1.25 eq., 106 mg, 0.868 mmol) were dissolved in dry

CH₂C1₂ (50 mL) and the mixture was refluxed under N₂ for 24 h. Extraction of the mixture with water (2 x 50 mL), aqueous HBr (0.1 M, 2 x 50 mL), and water (2 x 50 mL), drying over anhydrous Na₂S0₄ and concentration gave a residue, which, after flash-CC on silica with a toluene- acetone gradient, gave 10-6 (377 mg, 92%). TLC (toluene/acetone/MeOH 6/1/1 v/v): R_f = 0.68. UV/Vis (CH₂C1₂) : A_MAX = 370 nm. H NMR: retinoyl part:

7.072 (m, 1H, H-C7), 6.325 (s, 1H, H-C8), 6.291 (s, 1H, H-C10), 6.181 (s, 1H, H-Cl 1), 6.153-6.138 (d, 1H, J = 6 Hz, H-C12), 5.832 (s, 1H, H-C14), 2.391 (s, 3H, H-C17), 2.103 (m, 2H, H-C4), 2.010 (s, 3H, H-C16), 1.665 (m, 2H, H-C3), 1.501 (m, 2H, H-C2), 1.051 (s, 6H, H-C18-19). Non retinoyl part:4.283 (dt, 2H, H-a), 4.333 (dt, 2H, H-b), 4.313 (dt, 2H, Η- ), 3.534 (dt, 2H, H-20, 4.078 (dt, 2H, H-l "), 1.687 (m, 2H, H-2"), 1.371 (m, 2H, H-3"), 1-301 (m, 2H, H-4"), 1.304 (m, 2H, H-5 ), 0.883 (t, 3H, H-6"). ¹³C NMR: retinoyl part : 167.5 (C=0, C-15), 154.012 (C, C-13), 139.886 (C, C-6), 137.717 (CH, C-8), 137.214 (C, C-

9) , 134.871 (CH, C-12), 131.456 (CH, C-11), 130.103 (C, C-5), 129.399 (CH, C-

10) , 128.881 (CH, C-7), 117.656 (CH, C-14), 39.604 (CH₂, C-2), 33.721 (CH₂, C-4), 33.114 (C, C-l), 28.951 (2CH₃, C-18-19), 21.743 (CH₃, C-20), 19.211 (CH₂, C-3), 13.944 (CH₃, C-17), 12.924 (CH₃, C-17).

Non retinoyl part : 65.756 (d,

5.5 Hz, CH₂, C-a), 62.232 (d, 7.2 Hz, CH₂, C-b), 66.649 (d, ²J_C-_P = 5.5 Hz, CH₂, C-Γ), 29.391 (d, ³J_C__P = 7.2 Hz, CH₂, C-2^'), 68.459 (d, ²J_C-_P= 6.1 Hz, CH₂, C-l ^"), 30.175 (d, ³J_C-_P= 7.2 Hz, CH₂, C- 2^"), 25.061 ( CH₂, C-3^"), 31.303 (CH₂, C-4^"), 22.527 (CH₂, C-5 ^"), 13.999 (CH3-C-6 ^"). ³¹P NMR: - 0.866. HRMS (ESI): C₃₀H48BrO₆P calcd. 615.2445 (M+H) found 615.2467.

2-(( choline ) (hexyloxy)phosphoryloxy)ethoxy-retinoate ( C20-6)

10-6 (358 mg, 0.582 mmol), was dissolved in CHCl₃/iPrOH/DMF (3/5/5 v/v, 50 mL), NMe₃ (45% in water, 10 mL) was added and the mixture stirred at room temperature under N₂ for 4 days. Purification by flash-CC gave C20-6 (288 mg, 74%). TLC (CHCl₃/MeOH/H₂0 40:50:10 v/v): R_f = 0.28. UV/Vis (CH₂C1₂): A_max = 368 nm. ^XH NMR: retinoyl part as described under 10-6. 4.300 (dt, 2H, H-a), 4.350 (dt, 2H, H-b), 4.490 (dt, 2H, Η-Γ), 4.160 (dt, 2H, H-20, 3.500 (s, 9H, H- 47H-57H-60, 4.020 (dt, 2H, H-l ^"), 1.630 (m, 2H, H-2^"), 1.290 (m, 2H, H-3 ^"), 1.250 (m, 2H, H-4^"), 1.250 (m, 2H, H-5^"), 0.840 (t, 3H, H-6^"). ¹³ C NMR: retinoyl part as described under

10-6. 66.370 (d, ²J_C-_P= 5.40 Hz, CH₂, C-a), 62.910 (d, J_C-_P = 7.07 Hz, CH₂, C- b), 61.500 (d, ²Jc-_P= 5.40 Hz, CH₂, C-Γ), 65.360 (d, ³J_C-_P = 6.06 Hz, CH₂, C-20,

54.420 (CH₃, C-47C-57C-60, 68.980 (d, ²J_C-_P= 6.06 Hz, CH₂, C-l"), 30.220 (d, ³JC-P = 6.70 Hz, CH₂, C-2^' , 25.030 (d, ⁴J_c_p = 3.37 Hz, CH₂, C-3 ^"), 31.250 (CH₂, C-4^' , 22.490 (CH₂, C-5 ^"), 13.980 (CH₃, C-6^"). 31 P NMR: - 1.026. HRMS (ESI): C₃3H₅₇N0₆P calcd. 594.3924 (M⁺), found 594.3926.

2-((2-bromoethoxy) (tetradecyloxy) phosphoryloxyjethoxy-retionoate (10-14)

Retinoic acid (9) (200 mg, 0.666 mmol) and 4-14 (356 mg, 0.799 mmol) were reacted as described for 10-6 giving 10-14 (423 mg, 87%). TLC

(toluene/acetone/MeOH 6:1 :1 v/v): R_f = 0.80. UV/Vis (CH₂C1₂): _max = 369 nm . ^XH NMR: retinoyl part as described under 4-6. 4.288 (dt, 2H, H-a), 4.334 (dt, 2H, H-b), 4.313 (dt, 2H, Η-Γ), 3.536 (dt, 2H, H-2^'), 4.077 (dt, 2H, H-l ^"), 1.692 (m, 2H, H-2^' , 1.347 (m, 2H, H-3 ^"), 1.3-1.1 (16H, H-4^"-H-l l ^"), 1.265 (m, 2H, H-12^' , 1.268 (m, H-13 ^"), 0.883 (t, 3H, H-14^"). ¹³C NMR: retinoyl part as described under 10-6. 65.843 (d, ²J_C-P= 5.5 Hz, CH₂, C-a), 62.232 (d, ³J_C-P= 6.6 Hz, CH₂, C-b), 66.649 (d, ²J_C__P = 5.5 Hz, CH₂, C-Γ), 29.425 (d,

6.6 Hz, CH₂, C-2 , 68.459 (d, ²J_C-_P = 6.6 Hz, CH₂, C-l ^"), 30.219 (d, ³J_C-_P= 6.6 Hz, CH₂, C-2^'0, 25.398 (CH₂, C-3 ^"), 29.75-29.0 (8C, CH₂, C-4^"-C-l l ^"), 31.925 (CH₂-C-12^' , 22.695 (CH₂-C-13 ^"), 14.131 (CH₃-C-14^"). ³¹P NMR: -0.872. HRMS (ESI): C₃8H₆₄Br0₆P calcd. 727.3697 (M+H), found 727.3730.

2-((choline) (tetrcidecyloxy)phosphoryloxy)ethoxy-retinocite

-14)

10-14 (432 mg, 0.594 mmol) was reacted as described for C20-6 . Purification by flash-CC gave C20-14 (256 mg, 55%). TLC (CHCl₃/MeOH/H₂0 40:50:10 v/v): R_f = 0.38. UV/Vis (CH₂C1₂): _max = 371 nm. ^lE NMR: retinoyl part as described under 10-6. 4.319 (dt, 2H, H-a), 4.340 (dt, 2H, H-b), 4.541 (dt, 2H, Η- ), 4.232 (dt, 2H, H-20, 3.558 (s, 9H, H-47H-57H-6^'), 4.079dt, 2H, H-l ), 1.680 (m. 2H, H- 2"), 1.312 (m, 2H, H-3"), 1.3-1.1 (16H, Η-4"-Η-1Γ), 1.258 (m, 2H, H-12"), 1.261 (m, 2H, H-13 "), 0.879 (t, 3H, H-14"). ¹³C NMR: retinoyl part as described under 10-6. 66.535 (d, ²J_C-_P= 5.5 Hz, CH₂, C-a), 62.237 (d, ³J_C-_P= 6.6 Hz, CH₂, C-b), 61.585 (d, ²J_C._P= 5.5 Hz, CH₂, C-Γ), 65.623 (d, ³J_C._P= 6.6 Hz, CH₂, C-2 , 54.726 (CH₃, C-47C-57C-6^'), 69.183 (d, ²J_C._P= 6.6 Hz, CH₂, C- 1 ^"), 30.406 (d, ³Jc-_P= 6.6 Hz, CH₂, C-2^"), 25.586 (CH₂, C-3 ), 30.4-29.0 (8C, CH₂, C-4^"-C-l l ), 32.110 (CH₂, C-12^"), 22.810 (CH₂, C-13 ^"), 14.290 (CH₃, C-14^' . ¹P NMR: -1.095. HRMS (ESI):

C₄iH₇3N0₆P calcd. 706.5176 (M⁺), found 706.5187.

2-((2-bromoethoxy) (hexadecyloxy) phosphoryloxy)ethoxy-retionoate (10-16)

Retinoic acid (9) (515 mg, 1.7 mmol) and 4-16 (1.24 eq. 1 g, 2.10 mmol) were reacted as described for 10-6 giving 10-16 (990 mg, 69%). TLC

(toluene/acetone//MeOH 6/1/1 v/v): R_f = 0.61. UV/Vis (CH₂C1₂): A_max = 355 nm. ^XH NMR: retinoyl part as described under 10-6. 4.325 (dt, 2H, H-a), 4.334 (dt, 2H, H-b), 4.30 (dt, 2H, H-Cl ^'),

3.532 (dt, 2H, H-C2^'), 4.078 (dt, 2H, H-Cl ^"), 1-707 (m, 2H, H-C2 ), 1.471 (m, 2H, H-C3^"), 1.280-1.221 (20H, H-C4 -H-C13 ), 1-278 (m, 2H, H-C12 ), 1.246 (m, 2H, H-C13 ), 0.875 (t, 3H, H-C16"). ¹³C NMR: retinoyl part as described under 10-6.

65.776 (CH₂, C-a), 62.238 (CH₂, C-b), 66.665 (CH₂, C-l '), 29.392 (CH₂, C-2'), 68.411 (CH₂, C-l "), 30.211 (CH2, C-2"), 25.375 (CH₂, C-3"), 29.559-29.126 (IOC,

CH₂, C-4--C-13"), 31.903 (CH₂, C-14"), 22.670 (CH₂, C15"), 14.103 (CH₃, C16^"). ³¹P NMR : -0.811. HRMS (ESI): C₄oH₆₈Br0₆P calcd. 754.3920 (M+H), found 754.3921. -( ( choline) (hexadecyloxy)phosphoryloxy)ethoxy-retinoate ( C20-16)

10-16 (400 mg, 0.52 mmol) was reacted as described for C20-6 . Purification by flash-CC gave C20-16 (450 mg, 99%). TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v): R_f = 0.81. UV/Vis (CH₂C1₂): _max = 456 nm. ^XH NMR: retinoyl part as described under 10-6. 4.147 (dt, 2H, H-a), 4.291 (dt, 2H, H-b), 4.531 (dt, 2H, H-Cl '), 4.146 (dt, 2H, H-C2'), 3.486 (s, 9H, H-C4', H-C5', H-C6 , 4.050 (dt, 2H, H- Cl ^' , 1.662 (m, 2H, H-C2^"), 1.242 (m, 2H, H-C3^"), 1.288-1.190 (20H, H-C4, H-C13^"), 1.215 (m, 2H, H-C14^"), 1.210 (m, 2H, H-C15 ^"), 0.862 (t, 3H, H- C16^"). ^"). ¹ C NMR: retinoyl part as described under 10-6. 66.322 (CH₂, C-a), 62.050 (CH₂, C-b), 61.615 (CH₂, C-Γ), 65.566 (CH₂, C-2^'), 54.423 (CH₃, C-4^', C-5 ^', C-6 , 69.011 (CH₂, C-l^"), 30.242 (CH₂, C-2^"), 25.344 (CH₂, C-3^"), 30.242-29.144 (IOC, CH₂, C-4^"-C-l l^"),

31.887 (CH₂, C-14"), 22.654 (CH₂, C-15"), 14.093 (CH₃, C-16"). ³¹P NMR: - 1.081

HRMS (ESI): C₄₃H₇₇N0₆P calcd. 734.5476 (M⁺), found 734.5473.

2-((2-bromoethoxy) (octadecyloxy) phosphoryloxy)ethoxy-retionoate (10-18)

Retinoic acid (9) (500 mg, 1.6 mmol) and 4-18 (1.24 eq. 1 g, 2.10 mmol) were reacted as described for 10-6 giving 10-18 (949 mg, 75%). TLC

(toluene/acetone/VMeOH 6/1/1 v/v): R_f = 0.65. UV/Vis (CH₂C1₂): 2_max = 355 nm. ^XH NMR: retinoyl part as described under 10-6. 4.296 (dt, 2H, H-a), 4.341 (dt, 2H, H-b), 4.306 (dt, 2H, H-Cl ^'), 3.550 (dt, 2H, H-C2^'), 4.087 (dt, 2H, H-Cl^"), 1.713 (m, 2H, H-C2^"), 1.422 (m, H-C3^"), 1.3-1.1 (24H, H-C4^", H-C15^"), 1.259 (m, 2H, H-C16^"), 1-271 (m, 2H, H-C17^"), 0.892 (t, 3H, H-C18^"). ¹³C NMR: retinoyl part as described under 10-6. 65.823 (CH₂, C-a), 62.243 (CH₂, C-b), 66.661 (CH₂, C-l ^'), 29.424 (CH₂, C-20, 68.456 (CH₂, C-l ^"), 30.231 (CH₂, C-2^"), 25.441 (C2, C-3^"), 29.56-29.10 (12C, CH₂, C4"-C15"), 31.923 (CH₂, C-16"), 22.731 (CH₂, C-17"), 14.122 (CH₃, C- 18")· ³¹P NMR: -0.621. HRMS (ESI): C₄₂H72Br0₆P calcd. 782.4245 (M⁺), found 782.4245. -((choline) (octadecyloxy)phosphoryloxy)ethoxy-retinoate (C20-18)

10-18 (350 mg, 0.44 mmol) was reacted as described for C20-6. Purification by flash-CC gave C20-18 (300 mg, 81%). TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v): R_f = 0.84. UV/Vis (CH₂C1₂): _max = 455 nm. ^XH NMR: retinoyl part as described under 10-6. ^!H NMR: retinoyl part as described under 10-6. 4.315 (dt, 2H, H-a), 4.347 (dt, 2H, H-b), 4.546 (dt, 2H, H-Cl '), 4.098 (dt, 2H, H-C2'), 3.505 (s, 9H, H-C4', H-C5', H-C6'), 4.080 (dt, 2H, H-₂C1 "), 1.694 (m, 2H, H-C2"), 1-243 (m, 2H, H-C3"), 1.331-1.225 (24H, H-4C, H-C15 "), 1-221 (m, 2H, H-C16"), 1.210 (m, 2H, H-C17"), 0.891 (t, 3H, H-C18"). ¹³C NMR: : retinoyl part as described under 10-6. 66.619 (CH₂, C-a), 62.231 (CH₂, C-b), 61.615 (CH₂, C- Γ), 65.779 (CH₂, C-20, 54.617 (CH₃, C-4^', C-5^', C-6^'), 69.042 (CH₂, C-1'0, 30.238 (CH₂, C-2^'0, 25.386(CH₂, C-3^'0, 29.73-29.48 (12C, CH₂, C-4^", C- 11 ^"), 31.918 (CH, C-16^"), 22.685 (CH₂, C-17^"), 14.116 (CH₃, C-18^"). 31P NMR: -1.112. HRMS (ESI): C₄₅H₈iN0₆P calcd. 762.5781 (M⁺), found

762.5779.

2-((2-bromoethoxy) (icosyloxy) phosphoryloxy) ethoxy-retinoate

-20).

Retinoic acid (9) (200 mg, 0.666 mmol) and 4-20 (423 mg, 0.799 mmol) were reacted as described for 10-6 giving 10-20 (478 mg, 88%). TLC:

(toluene/acetone/ MeOH 6:1:1 v/v): R_f = 0.85. UV/Vis (CH₂C1₂): X_mm = 370 nm. ^XH NMR:

retinoyl part as described under 10-6. 4.283 (dt, 2H, H-a), 4.333 (dt, 2H, H-b), 4.308 (dt, 2H, Η- ), 3.550 (dt, 2H, H-2 , 4.075 (dt, 2H, H-l "), 1.692 (m, 2H, H-2"), 1.344 (, 2H, H-3 , 1.35-1.15 (28H, H-4"-H-17"), 1.259 (m, 2H, H- 18"), 1.277 (m, 2H, H-19"), 0.885 (t, 3H, H-20"), ¹³C NMR: retinoyl part as described under 10-6. 65.673 (d, ²J_C-_P= 5.5 Hz, CH₂, C-a), 62.239 (³J_C-_P= 6.6 Hz, CH₂, C-b), 66.649 (²J_C__P= 5.5 Hz, CH₂, C-Γ), 29.945 ( J_C-_P= 6.6 Hz, CH₂, C-2 , 68.466 (d, ²J_C._P= 6.6 Hz, CH₂, C-l ^"), 30.219 (d, ³J_C._P = 6.6 Hz, CH₂, C- 2^"), 25.406 (CH₂, C-3^"), 29.8-29.0 (14C, CH₂, C-4^"-C-17^"), 31.933 (CH₂, C- 18^"), 22.702 (CH₂, C-19^"), 14.131 (CH₃, C-20^"). ¹P NMR: -0.871. HRMS (ESI): C44H₇₅Br0₆P calcd.811.4636 (M+H), found 811.4668.

2-(( choline icosyloxy)phosphoryloxy)ethoxy-retinoate ( C20-20 ).

10-20 (460 mg, 0.566 mmol), was reacted as described for for C20-6.

Purification by flash-CC gave C20-20 (362 mg, 74%). TLC (CHCl₃/MeOH/H₂0 40:50:10 v/v): R_f = 0.42 UV/Vis (CH₂C1₂): l_max = 369 nm. ^lR NMR: retinoyl part as described under

4-6. 4.321 (dt, 2H, H-a), 4.337 (dt, 2H, H-b), 4.552 (dt, 2H, Η- ), 4.226 (dt, 2H, H-2^'), 3.563 (s, 9H, H-47H-57H-60, 4.078 (dt, 2H, H-l ^"), 1.679 (m, 2H, H- 2"), 1.309 (m, 2H, H-3"), 1.4-1.1 (28H, H-4"/H-17"), 1.260 (m, 2H, H-18"), 1.266 (m, 2H, H-19"), 0.878 (t, 3H, H-20"). ¹³C NMR: retinoyl part as described under 4-6. 66.422 (d, ²J_C-_P= 5.5 Hz, CH₂, C-a), 62.107 (d, J_C-_P= 6.6 Hz, CH₂, C-b), 61.465 (d,

²Jc-_P= 5.5 Hz, CH₂, C-r), 65.459 (d, J_C-_P= 7.2 Hz, CH₂, C-20, 54.546 (CH3, C-47C-57C-6 , 69.026 (d, ²J_C-_P= 6.6 Hz, CH₂, C-l "), 30.246 (d, ³J_C-_P= 6.6 Hz, CH₂, C-2"), 25.459 (CH₂, C-3"), 30.3-28.6 (14C, C-4"/C-17"), 31.939 (CH₂, C18"), 22.692 (CH₂, C-19"), 14.139 (CH₃, C-20"). ¹P NMR: -1.153. HRMS (ESI): C47H₈₅N0₆P calcd.790.6115 (M⁺), found 790.6125. -Apo-8'-carotenoic acid

C29

Hydrolysis of P-apo-8 '-carotenoate gave P-Apo-8'-carotenoic acid in 90% yield. H NMR: in accordance with Ref. ^[-^] -((2-Bromoethoxy)(hexyloxy)phosphoryloxy)ethoxy-^-apo-8'-carotenoate (5-6)

β-Αρο-8 -caiotenoic acid (300 mg, 0.694 mmol), 4-6 (1.2 eq., 257 mg, 0.773 mmol), chlorotripjTrolidinophosphonium hexafluorophosphate (PyCloP, 1.25 eq., 349 mg, 0.868 mmol), N-ethyl diisopropylamine (DIEA, 0.65 eq., 58 mg, 0.451 mmol), and DMAP (1.25 eq., 106 mg, 0.868 mmol) were dissolved in dry CH2CI2 (50 mL) and the mixture was refluxed under Ν2 for 24 h. Extraction of the mixture with water (2 x 50 mL), aqueous HBr (0.1 M, 2 x 50 mL), and water (2 x 50 mL), drying over anhydrous Na₂S0₄ and concentration gave a residue, which, after flash-CC on silica with a toluene- acetone gradient, gave 5-6 (322.3 mg, 62%). TLC (toluene/acetone/MeOH 6:1 :1 v/v): R_f = 0.68. UV/Vis (CH₂C1₂): ax = 453 nm. ^XH NMR: carotenoyl portion in accordance with β-Αρο-8'- carotenoic acid, 4.330 (dt, 2H, H-l), 4.390 (dt, 2H, H-2), 4.290 (dt, 2Η,Η-Γ), 3.540 (dt, 2H, H-2^'), 4.080 (dt, 2H, H-l ^"), 1.690 (m, 2H, H-2^"), 1.380 (m, 2H, H-3 ^"), 1.310 (m, 2H, H-4^"), 1.320 (m, 2H, H-5 ^"), 0.880 (t, 3H, H-6^"). ¹³C NMR: carotenoyl portion is in accordance with -Apo-8'-carotenoic acid, 65.740 (d, ²JC-P = 5.60 Hz, CH₂, C-l), 63.050 (d, J_C-_P = 7.10 Hz, CH₂, C-2^'), 66.650 (d, ²Jc-p = 5.10 Hz, CH₂, C-l 0, 29.570 (d, J_C-_P = 7.60 Hz, CH₂, C-2^'), 68.450 (d, ²Jc-p = 6.10 Hz, CH₂, C-l ^"), 30.170 (d, ³J_C-p = 12.20 Hz, CH₂, C-2^"), 25.060 (d, ⁴Jc-p = 4.06 Hz, CH₂, C-3 ^"), 31.280 (CH₂, C-4^"), 22.510 (CH₂, C-5 ^"), 13.980 (CH₃, C-6^"). ³¹P NMR: -0.879. MS (EI): 746.85 + 748.85 (1 : 1, M⁺); HRMS (EI): C4oH₆oBr0₆P calcd. 748.32993 (M ⁺), found 748.33023. 2-( ( Choline)(hexyloxy)phosphoryloxy)ethoxy^-apo-8'-carotenoate (C30-6)

Carotenoate 5-6 (322 mg, 0.43 mmol) was dissolved in CHCl₃/iPrOH/DMF (3/5/5 v/v, 50 mL), NMe₃ (45% in water, 10 mL) was added and the mixture stirred at room temperature under N₂ for 4 days. Flash-CC on neutral A1₂0₃ gave C30-6 (264 mg, 77%). TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v): R_f = 0.08.

UV/Vis (CH₂C1₂): / ax = 458 nm. ^lE NMR: carotenoyl part in accordance with 2, 4.300 (dt, 2H, H-l), 4.350 (dt, 2H, H-2), 4.490 (dt, 2Η,Η-Γ), 4.160 (dt, 2H, H-2^'), 3.500 (s, 9H, H-47H-57H-6^'), 4.020 (dt, 2H, H-l ^"), 1.630 (m, 2H, H- 2^"), 1.290 (m, 2H, H-3 ^"), 1.250 (m, 2H, H-4^"), 1.250 (m, 2H, H-5 ^"), 0.840 (t,

13 2

3H, H-6^"). C NMR: carotenoyl portion is in accordance with 2, 66.370 (d, Jc- P = 5.40 Hz, C¾, C-l), 62.910 (d, ³J_C-_P = 7.07 Hz, CH₂, C-2), 61.500 (d, ²J_C-_P = 5.40 Hz, CH₂, C-l '), 65.360 (d, ³J_C__P = 6.06 Hz, CH₂, C-2'), 54.420 (C¾, C- 47C-57C-6^'), 68.980 (d, ²J_C-P = 6.06 Hz, CH₂, C-l ^"), 30.220 (d, ³J_C-_P = 6.70 Hz, CH₂, C-2"), 25.030 (d, ⁴J_C._P = 3.37 Hz, CH₂, C-3 "), 31.250 (CH₂, C-4"), 22.490 (CH₂, C-5 "), 13.980 (CH₃, C-6"). ³¹P NMR: -1.026. HRMS ^[-^]:

C₄3H6₉ 0₆P calcd. 726.4803 (M⁺), found 726.4869.

2-((2-bromoethoxy)(dodecyloxy)phosphoryloxy)ethoxy-fi-apo-8'-carotenoate (5-

β-Αρο-8 -carotenoic acid (2) (200 mg, 0.459 mmol), and 4-12 (1.2 eq., 230 mg, 0.550 mmol), were reacted as described for 5-6 giving 5-12 (200.7 mg, 53%). TLC (hexane/acetone 8:2 v/v): R_f = 0.30. UV/Vis (CH₂C1₂): /._mx = 457 nm. ^lU NMR: C30:9 part is in accordance with 2, 4.330 (dt, 2H, H-l), 4.440 (dt, 2H, H- 2), 4.320 (dt, 2Η,Η-Γ), 3.540 (dt, 2H, H-2'), 4.090 (dt, 2H, H-l "), 1.700 (m, 2H, H-2"), 1.370 (m, 2H, H-3 "), 1.30-1.25 (m, 12H, H-4"-H-9"), 1.260 (m, 2H, H-10^"), 1.300 (m, 2H, H-ll ^"), 0.890 (t, 3H, H-12^"). C NMR: C30:9 part is in accordance with 2, 65.750 (d, ²J_C-_P = 5.73 Hz, CH₂, C-l), 63.060 (d, ³J_C-_P = 6.40 Hz, CH₂, C-2), 66.670 (d, ²J_C-_P = 5.05 Hz, CH₂, C-l '), 29.350 (d, J_C-_P = 6.40 Hz, CH₂, C-2'), 68.480 (d, ²J_C-_P = 6.06 Hz, CH₂, C-l "), 30.220 (d, ³J_C-_P = 6.74 Hz, CH₂, C-2"), 25.400 (CH₂, C-3"), 29.150 (CH₂, C-4"), 29.520 (CH₂, C-5 "), 29.580 (CH₂, C-6"), 29.650 (CH₂, C-l^"), 29.630 (CH₂, C-8'0, 29.640 (CH₂, C-9"), 31.91 (CH₂, C-10"), 22. (CH₂, C-ll "), 14.130 (CH₃, C-12"). ³¹P NMR: -0.971. MS (EI): 830.1 + 832.2 (1:1, M⁺). HRMS (EI): C46H₇₂Br0₆P calcd. 830.42500 (M⁺), found 830.42748.

2-(( choline dodecyloxy)phosphoryloxy)ethoxy-$-apo-8 '-carotenoate (C30-12 )

5-12 (200.7 mg, 0.241 mmol) was reacted as decribed for C30-6. Purification by flash- CC gave C30-12 (130 mg, 61%). TLC (CHCl₃/MeOH/H₂0 70/30/3 v/v): R_f = 0.47. UV/Vis (CH₂C1₂): l_m5X = 458 nm. ^lH NMR: C30:9 part is in accordance with 2, 4.240 (dt, 2H, H-l), 4.270 (dt, 2H, H-2), 4.450 (dt, 2H, H- 1 , 4.010 (dt, 2H, H-20, 3.390 (s, 9H, H-47H-57H-60, 3.950 (dt, 2H, H-1 ^'0, 1.540 (m, 2H, H-2'0, 1.180 (m, 2H, H-3'0, 1.12-0.85 (m, 12H, H-4"-H-9'0, 1.120 (m, 2H, H-10'0, 1-150 (m, 2H, H- 11 "), 0.750 (t, 3H, H-12"). ¹ C NMR: C30:9 part is in accordance with 2, 66.370 (d, ²J_C-_P = 5.73 Hz, CH₂,C-1), 63.020 (d, ³Jc-p = 6.40 Hz, CH₂, C-2), 61.590 (d, ²J_C-p = 5.05 Hz, CH₂, C-10, 65.430 (d, ³Jc-p = 6.40 Hz, CH₂, C-20, 54.370 (CH₃, C-47C-57C-60, 68.930 (d, ²J_C-P = 6.06 Hz, CH₂, C-l^"), 30.250 (d, J_C-_P = 6.74 Hz, CH₂, C-2^"), 25.380 (CH₂, C- 3^"), 29.200 (CH₂, C- 4^"), 29.550 (CH₂, C-5 ^"), 29.590 (CH₂, C-6^"), 29.670 (CH₂, C-7^"), 29.620 (CH₂, C-8^'0, 29.640 (CH₂, C-9^"), 31.910 (CH₂, C-10^"), 22.680 (CH₂, C-ll^"), 13.950 (CH₃, C-12^"). ³¹P NMR: -1.121. HRMS ^[-^]: C4₉Hsi 0₆P calcd. (M⁺) 810.5802 (M⁺), found 810.5793. 2-((2-bromoethoxy)(tetradecyloxy)phosphoryloxy)ethoxy-fi-apo-8'-carotenoate -14)

β-Αρο-8 carotenoic acid (2) (300 mg, 0.694 mmol) and 4-14 (1.2 eq., 344 mg, 0.773 mmol) were reacted as described for 5-6 to 5-14 (425 mg, 71%). TLC (hexane/acetone 8:2 v/v): R_f = 0.27. UV/Vis (CH₂C1₂): ^_max = 457 nm. ^XH NMR: C30:9 part is in accordance with 2, 4.288 (dt, 2H, H-l), 4.334 (dt, 2H, H-2), 4.313 (dt, 2H, Η-Γ), 3.536 (dt, 2H, H-2'), 4.077 (dt, 2H, H-l "), 1.692 (m, 2H, H-2^"), 1.347 (m, 2H, H-3 ^"), 1.3-1.1 (16H, H-4"-H-l l ^"), 1.265 (m, 2H, H- 12^"), 1.268 (m, 2H, H-13 ^"), 0.883 (t, 3H, H-14^"). ¹³C NMR: C30:9 part is in accordance with 2, 65.843 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l), 62.232 (d, ³J_C-P = 6.6 Hz, CH₂, C-2), 66.649 (d, ²J_C._P = 5.5 Hz, CH₂, C-Γ), 29.425 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^'), 68.459 (d, ²J_C-_P = 6.6 Hz, CH₂, C-l ^"), 30.219 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^"), 25.398 (CH₂, C-3 ^"), 29.75-29.0 (8C, CH₂, C-4^"-C-l l ^"), 31.925 (CH₂, C-12^"), 22.695 (CH₂, C-13^"), 14.131 (C¾, C-14^"). ³¹P NMR: -0.872.

2-(( choline)( tetradecyloxy)phosphoryloxy)ethoxy-fi-apo-8 '- carotenoate ( C30- 14)

5-14 (400 mg, 0.465 mmol) was reacted as described for C30-6. Purification by flash-CC gave C30-14 (242 mg, 57%).TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v): R_f = 0.68. UV/Vis (CH₂C1₂): 2_max = 461 nm. ^XH NMR: C30:9 part is in accordance with 2, 4.335 (dt, 2H, H-l), 4.365 (dt, 2H, H-2), 4.532 (dt, 2H, Η- ), 4.139 (dt, 2H, H-2'), 3.497 (s, 9H, H-47H-57H-6'), 4.041 (dt, 2H, H-l "), 1.642 (m, 2H, H-2"), 1.273 (m, 2H, H-3 "), 1.3-1.1 (16H, H-4-H-11 "), 1.222 (m, 2H, H-12"), 1.227 (m, 2H, H-13 "), 0.845 (t, 3H, H-14"). ¹³C NMR: C30:9 part is in accordance with 2, 66.444 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l), 63.091 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2), 61.605 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l '), 65.569 (d, ³J_C-_P = 7.2 Hz, CH₂, C-2^'), 54.579 (CH₃, C-47C-57C-6^'), 69.096 (d, ²J_C-_P = 6.6 Hz, CH₂, C- r , 30.379 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2^"), 25.508 (CH₂, C-3 ^"), 30.3-28.8 (8C, CH₂, C-4^"-C-l l ^"), 31.991 (CH₂, C-12^"), 22.774 (CH₂, C-13 ^"), 14.259 (CH₃, C-14' . ³¹P NMR: -1.101. HRMS ^[-^]: C₅iH₈₅N0₆P calcd. 838.6115 (M⁺), found 838.6121.

2-((2-bromoethoxy)(hexadecyloxy)phosphoryloxy)ethoxy-fi-apo-8'- carotenoate -W

β-Αρο-8 -carotenoic acid (2) (450 mg, 1.04 mmol) and 4-16 (2.39 eq., 1.174 mg, 2.48 mmol) were reacted as described for 5-6 giving 5-16 (631 mg, 68%). TLC (toluene/acetone/methanol 6:1 :1 v/v): R_f = 0.80. UV/Vis (CH₂C1₂): 2_max = 455 nm. ^lU NMR: C30:9 part is in accordance with 2, 4.284 (dt, 2H, H-1), 4.344 (dt, 2H, H-2), 4.309 (dt, 2H, Η- ), 3.536 (dt, 2H, H-2'), 4.091 (dt, 2H, H-1 "), 1.712 (m, 2H, H-2"), 1.412 (m, 2H, H-3"), 1.3-1.1 (20H, H-4"-H-13 "), 1.261 (m, 2H, H-12"), 1.265 (m, 2H, H-13 "), 0.893 (t, 3H, H-16"). ¹³C NMR: C30:9 part is in accordance with 2, 65.833 (CH₂, C-l), 62.241 (CH₂, C-2), 66.651 (CH₂, C- Γ), 29.426 (CH₂, C-20, 68.461 (CH₂, C-l "), 30.221 (CH₂, C-2"), 25.431 (CH₂, C-3 "), 29.77-29.2 (IOC, CH₂, C-4"-C-13"), 31.922 (CH₂, C-14"), 22.721 (CH₂, C-15"), 14.132 (CH₃, C-16"). ¹P NMR: -0.601. HRMS: CsoHsoBrOeP calcd. 886.4869 (M+H), found 886.4869.

2-((choline)(hexadecyloxy)phosphoryloxy)ethoxy-^>-apo-8'-carotenoate (C30-16)

5-16 (200 mg, 0.225 mmol) was reacted as described for C30-6. Purification by flash-CC gave C30-16 (155 mg, 72%). TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v): R_f = 0.62. UV/Vis (CH₂C1₂): _max = 456 nm. H NMR: C30:9 part is in accordance with 2, 4.333 (dt, 2H, H-1), 4.355 (dt, 2H, H-2), 4.543 (dt, 2H, H- Γ), 4.123 (dt, 2H, H-2^'), 3.550 (s, 9H, H-4 H-57H-6^'), 4.032 (dt, 2H, H-1 "), 1.651 (m, 2H, H-2"), 1.243 (m, 2H, H-3"), 1.3-1.1 (20H, H-4-H-13"), 1.212 (m, 2H, H-14"), 1.217 (m, 2H, H-15"), 0.815 (t, 3H, H-16"). ¹³C NMR: C30:9 part is in accordance with 2, 66.414 (CH₂, C-1), 63.081 (CH₂, C-2), 61.615 (CH₂, C-1 , 65.529 (CH₂, C-2^'), 54.559 (C¾, C-4 C-57C-6^'), 69.186 (CH₂, C- 1 "), 30.369 (CH₂, C-2"), 25.508 (CH₂, C-3"), 30.2-28.5 (IOC, CH₂, C-4"-C- 11 "), 31.981 (CH₂, C-14"), 22.764 (CH₂, C-15"), 14.289 (CH₃, C-16"). ¹P NMR: -1.121. HRMS: C₅₃H₈₉N0₆P calcd. 866.6422 (M⁺), found 866.6423.

2-((2-bromoethoxy)(octadecyloxy)phosphoryloxy)ethoxy-fi-apo-8'- carotenoate (5-18)

β-Αρο-8 carotenoic acid (2) (670 mg, 1.54 mmol), 4-18 (2.39 eq., 1856 mg, 3.70 mmol) were reacted as described for 5-6 giving 5-18 (858 mg, 61%). TLC (toluene/acetone/methanol 6:1 :1 v/v): R_f = 0.75. UV/Vis (CH₂C1₂): Λ™χ = 454 nm. *H NMR: C30:9part is in accordance with 2, 4.296 (dt, 2H, H-1), 4.341 (dt, 2H, H-2), 4.306 (dt, 2H, Η-Γ), 3.550 (dt, 2H, H-2'), 4.087 (dt, 2H, H-1 "), 1.713 (m, 2H, H-2"), 1.422 (m, 2H, H-3"), 1.3-1.1 (24H, H-4"-H-15 "), 1.259 (m, 2H, H-16"), 1.271 (m, 2H, H-17"), 0.892 (t, 3H, H-18"). ¹³C NMR: C30:9 part is in accordance with 2, 65.823 (CH₂, C-1), 62.243 (CH₂, C-2), 66.661 (CH₂, C- 1 ^'), 29.424 (CH₂, C-2^'), 68.456 (CH₂, C-1 "), 30.231 (CH₂, C-2"), 25.441 (CH₂, C-3 "), 29.56-29.1 (12C, CH₂, C-4"-C-15"), 31.943 (CH₂, C-16"), 22.731 (CH₂, C-17"), 14.122 (CH₃, C-18"). ¹P NMR: -0.621. HRMS: C₅₂H₈₄Br0₆P calcd. 914.5196 (M⁺), found 914.5189. -(( choline)(octadecyloxy)phosphoryloxy)ethoxy- -apo-8 '-carotenoate ( C30-18 )

5-18 (240 mg, 0.261 mmol) was reacted as described for C30-6. Purification by flash-CC gave C30-18 (200 mg, 78%). TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v): R_f = 0.82. UV/Vis (CH₂C1₂): _max = 455 nm. ^lH NMR: C30:9 part is in accordance with 2, 4.313 (dt, 2H, H-l), 4.345 (dt, 2H, H-2), 4.553 (dt, 2H, H- 1 , 4.133 (dt, 2H, H-20, 3.551 (s, 9H, H-47H-57H-60, 4.042 (dt, 2H, H-l "), 1.641 (m, 2H, H-2"), 1.253 (m, 2H, H-3"), 1.3-1.1 (24H, H-4-H-15"), 1.232 (m, 2H, H-16"), 1.216 (m, 2H, H-17"), 0.825 (t, 3H, H-18"). ¹³C NMR: C30:9 part is in accordance with 2, 66.434 (CH₂, C-l), 63.141 (CH₂, C-2), 61.625 (CH₂, C-10, 65.522 (CH₂, C-20, 54.565 (CH₃, C-47C-57C-60, 69.177 (CH₂, C- 1 "), 30.354 (CH₂, C-2"), 25.512 (CH₂, C-3"), 30.0-28.1 (12C, CH₂, C-4"-C- 11 "), 31.951 (CH₂, C-16"), 22.784 (CH₂, C-17"), 14.279 (CH₃, C-18"). ¹P NMR: -1.132. HRMS: C55H93NO6P calcd. 894.6729 (M⁺), found 894.6728.

2-((2-bromoethoxy)(icosyloxy)phosphoryloxy)ethoxy-fi-apo-8'- carotenoate (5-

β-Αρο-8 carotenoic acid (2) (397 mg, 0.919 mmol) and 4-20 (340 mg, 0.584 mmol) were reacted as described for 5-6 giving 5-20 (333 mg, 62%). TLC

(hexane/acetone 8:2 v/v): R_f = 0.28. UV/Vis (CH₂C1₂): _mm = 458 nm. ^lU NMR: C30:9 part in accordance with 2. 4.380 (dt, 2H, H-2), 4.313 (dt, 2H, Η-Γ), 3.535 (dt, 2H, H-20, 4.080 (dt, 2H, H-1 ^'0, 1.683 (dt, 2H, H-2^'0, 1.347 (dt, 2H, H-3 ^'0, 1.3-1.1 (28H, H-4"- H-17'0, 1.256 (m, 2H, H-18'0, 1.256 (m, 2H, H-19'0, 0.879 (t, 3H, H-20"), ¹³C NMR: C30:9 part is in accordance with 2, 65.778 (d, ²JC-P = 5.5 Hz, CH₂, C-l), 63.096 (d, ³J_C-_P = 6.6 Hz, CH₂, C-2), 66.693 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l 0, 29.457 (d, ³J_C._P = 6.6 Hz, CH₂, C-20, 68.510 (d, ²J_C-_P = 6.6 Hz, CH₂, C-l ^"), 30.241 (d, J_C-P = 6.6 Hz, CH₂, C-2^"), 25.420 (CH₂, C-3 ^"), 29.8-29.0 (14C, CH₂, C-4^"-C-17^"), 31.947 (CH₂, C-18^"), 22.717 (CH₂, C- 19^"), 14.146 (CH₃, C-20^"). ³¹P NMR: - 0.854. HRMS (ESI): Cs^sBrOeP calcd. 945.5568, found 945.5547 (M+H). 2-(( choline)(icosyloxy)phosphoryloxy)ethoxy-[j-apo-8 '-carotenoate ( C30-20 ) '

5-20 (300 mg, 0.317 mmol) was reacted as described for C30-6. Purification by flash-CC gave C30-20 (303 mg, 95%). TLC (CHCl₃/MeOH/H₂0 70:30:3 v/v) R_f = 0.78. UV/Vis (CH₂C1₂): ληαχ = 459 nm. ^XH NMR: C30:9 part in accordance with 2. 4.375 (dt, 2H, H-1), 4.403 (dt, 2H, H-2), 4.576 (dt, 2H, Η-Γ), 4.196 (dt, 2H, H-2^'), 3.549 (s, 9H, H-47H-57H-6^'), 4.073 (dt, 2H, H-1 ^"), 1.673 (m, 2H, H-2^"), 1.305 (m, 2H, H-3^"), 1.4-1.1 (28H, H-4^"-H-17^"), 1.260 (m, 2H, H- 18"), 1.262 (m, 2H, H-19"), 0.881 (t, 3H, H-20"). ¹³C NMR: C30:9 part is in accordance with 2, 66.530 (d, ²J_C-_P = 5.5 Hz, CH₂, C-l), 63.149 (d, ³J_C-_P = 6.6

6 0/ 1 Hz, CH₂, C-2), 61.662 (d, ²J_C-_P = 5.5 Hz, CH₂, C-Γ), 65.583 (d, ³J_C-_P = 7.2 Hz, CH₂, C-2^'), 54.530 (CH₃, C-47C-57C-6^'), 69.132 (d, ²J_C-_P = 6.6 Hz, CH₂, C- \ ^"), 30.418 (d, ³JC-P = 6.6 Hz, CH₂, C-2^"), 25.584 (CH₂, C-3 ^"), 30.4-28.7 (14C, CH₂, C-4^"-C-17' , 32.124 (CH₂, C-18 ^"), 22.821 (CH₂, C-19^"), 14.330 (CH₃, C-20' . ³¹ P NMR: -1.147. HRMS (ESI): C57H97NO6P calcd. 922.7054, found 922.7048 (M⁺).

2-(N,N-dimethylamino)ethyl-fi-apo-8 '-carotenoate (8)

P-Apo-8'-carotenoic acid (580 mg, 1.34 mmol) and 1,1 '-carbonyldi( 1 ,2,4- triazole) (275 mg, 1.68 mmol) were dissolved in dry CH₂C1₂ (75 mL) and a crystal of 4-(N,N-dimethylamino) pyridine (DMAP) was added. The mixture was stirred at room temperature under N₂ for 1 h, until TLC indicated full conversion. Dry 2-N,N-dimethylaminoethanol (0.82 mL, 8.19 mmol) was added, and the mixture was refluxed for 5 h. The mixture was washed with distilled water (3x50 mL), the organic phase dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by flash-CC on silica with

CH₂Cl₂/MeOH gradient and recrystallized from acetone (458 mg, 68%). TLC (toluene/acetone/MeOH, 6/1/1 v/v): R_f = 0.50. m.p.: 130.3-131.5 °C. UV/Vis (CH₂C1₂): = 457 nm. ^-NMR: Carotenoyl part in accordance with our previous work [23], 4.276 (t, 2H, H-1), 2.645 (t, 2H, H-2), 2.318 (s, 6H, H-4/H- Γ). ¹³C NMR: 62.696 (CH₂, C-l), 57.925 (CH₂, C-2), 45.879 (CH₃, C4/C-1 . HRMS (ESI): C₃₄H₄₉N0₂ calcd. 503.3763 (M+), found 503.3749.

2-(N,N-dimethylamino)ethyl-fi-apo-& '-carotenoate hydrobromide (1)

The dimethylamine analogue 8 (458.5 mg, 0.91 mmol) was dissolved in MeOH (30 mL) and HBr (1 M in water, 2 eq, 1.82 mL, 1.82 mmol) was added. The mixture was stirred at room temperature under N₂ for 3 h. The solvent was removed under reduced pressure and water by freeze-drying. The residue was dissolved in CH₂C1₂ and crystallized in hexane giving 1 (445.8 mg, 0.76 mmol, 84%). m.p.: 162.1-164.3 °C. UV/Vis (CH₂C1₂): _max = 462 nm. 1H NMR: 4.541 (m, 2H, H-1), 3.568 (m, 2H, H-2), 2.995 9s, 6H, H-4/H-1 '). ¹³C-NMR: 57.653 (CH₂, C-l), 55.698 (CH₂, C-2), 42.329 (CH₃, C4/C-1 . HRMS (ESI):

C₃₄H₅₀NO₂ calcd. 504.3842 (M+), found 504.3817.

61 2-bromoethyl-fi-apo-carotenoate (9)

Carotenoic acid 7 (1.00 g, 2.31 mmol), dicyclohexylcarbodiimide (DCC, 0.72 g, 3.47 mmol), DMAP (56 mg, 0.46 mmol) and 2-bromoethanol (5.77 g, 46.2 mol) were dissolved in dry CH₂CI₂ and stirred at room temperature under N₂ for 18 h. The reaction mixture was extracted with water (3 x 50 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was dissolved in cold acetone (10 mL) and filtered to remove the urea formed from DCC. The bromoethyl carotenoate 9 was recrystallized from acetone (1.06 g, 85 %). TLC

(hexane/acetone 8/2 v/v): R_f = 0.80. m.p. : 132.8-133.6 °C. UV/Vis (CH₂C1₂) :

= 460 ran. ^-NMR: 4.461 (t, 2H, H- l), 3.579 (t, 2H, H-2). ¹³C-NMR:

63.873 (CH₂, C-l), 29.096 (CH₂, C-2). HRMS (EI): C₃₂H₄₃Br0₂ calcd.

538.24463 (M⁺), found 538.24465.

2-(N,N,N-trimethyl) ethyl-fi-apo-8 '-carotenoate bromide (2)

Bromoethyl carotenoate 9 (600 mg, 1.11 mmol) was dissolved in

CHCls/iPrOH/DMF (3/5/5/ v/v 50 mL) and NMe₃ (45% in water, 5 mL) was added. The mixture was stirred at room temperature under N₂ for 4 days. The solvents were removed under reduced pressure, the residue dissolved in CH₂C1₂ (50 mL) and extracted with water (3 x 50 mL). The organic phase was dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by flash-CC A1₂C>3 with a toluene/acetone/MeOH gradient and the product was isolated with 5 % MeOH in acetone. The desired quaternized amine 2 was recrystallized from acetone (565 mg, 86%). TLC: (CHCl₃/MeOH H₂0, 65/25/4 v/v). R_f = 0.30. m.p. : 230.8-231.3 °C. UV/Vis (CH₂C1₂): λ_ΜΧ = 464 ran. ^-NMR: 4.670 ((b, 2H, H- l), 4.176 (b, 2H, H-2), 3.582 (s, 9H, H-4/H-5/H-6). ¹³C-NMR: 57.929 (CH₂, C- l), 65.131 (CH₂, C-2), 54.405 (CH₃, C-4/C-5/C-6). HRMS (ESI):

C₃₅H₅₂N0₂ calcd. 518.3998 (M⁺), found 518.3988.

2-(N-(2-hydroxyethyl), N-methyl-amino)ethyl-fi-apo-& '-carotenoate (10)

Carotenoic acid 7 (390 mg, 0.90 mmol), DCC (280 mg, 1.25 mmol) and DMAP (22 mg, 0.18 mmol) were dissolved in dry CH₂C1₂ (5mL) and N-methyl diethanolamine (5 mL) was added. The mixture was stirred at room temperature under N₂ for 3 days. The solution was extracted with water (3 x 50 mL), the organic layer dried over anhydrous Na₂S0₄ and concentrated in cold acetone (5 mL) and filtered to remove urea. Purification by flash-CC on silica with a

62 toluene/acetone gradient eluted the product at 10% acetone. The desired ethanolamine product 10 was recrystallized from acetone (197 mg, 41%). TLC (hexane/acetone 8/2 v/v): R_f = 0.38. UV/Vis (CH₂C1₂): = 456 ran. ^-NMR: 4.178 (t, 2H, H-l), 2.696 (t, 2H, H-2), 2.539 (t, 2H, H-4), 3.494 (t, 2H, H-5), 2.261 (s, 3H, H-l ^'). ¹³C-NMR: 62.005 (CH₂-C-1), 55.865 (CH₂, C-2), 58.891 (CH₂, C-4), 58.283 (CH₂, C-5), 42.008 (CH₃, C-V).

2-(^-(2-hydroxyethyl) ^,^-dimethyl-ammonium)ethyl-fi-apo- '-carotenoate iodide (3)

Ethanolamine 10 (190 mg, 0.35 mmol) was dissolved in dry THF (50 mL) and Mel (3 mL) was added. The mixture was stirred at room temperature under N₂ for 3 days and the crude product was isolated by filtration. The quaternized ethanolamine 3 was recrystallized from CH₂C1₂ and hexane (124 mg, 53%). TLC (CHCl₃/MeOH/H₂0 70/30/3 v/v): R_f = 0.37. m.p.: 183.2-185.7 °C. UV/Vis (CH₂C1₂): λ = 465 nm. ^-NMR: 4.655 (t, 2H, H-l), 4.097 (t, 2H, H-2), 3.882 (t, 2H, H-4), 4.169 ((t, 2H, H-5), 3.469 (s, 6H, H-17H-1 ^"). ¹³C-NMR: 58.036 (CH₂-C-1), 64.210 (CH₂, C-2), 66.640 (CH₂, C-4), 55.761 (CH₂, C-5), 53.142 (CH₃, C-17C-1 ^"). HRMS (ESI): C36H54NO3 calcd. 548.4102 (M⁺), found 548.4101.

2-(N,N-di(2-hydroxyethyl)-amino)ethyl-fi-apo-& '-carotenoate (11)

Carotenoic acid (1.216 g, 2.81 mmol), DCC (870 mg, 4.22 mmol) and DMAP (70 mg, 0.56 mmol) were dissolved in dry CH₂C1₂ (50 mL) and triethanolamine (10 mL) was added. The mixture was stirred at room temperature under N₂ for 3 days. The solution was extracted with water (3 x 50 mL), the organic layer dried over Na₂S0₄ and concentrated under reduced pressure. The residue was dissolved in cold acetone (5 mL) and filtered to remove DCC-urea. Purification by flash-CC on silica with a toluene/acetone gradient eluted the product at 10% acetone. Diethanolamine 11 was recrystallized from acetone (937 mg, 59%). TLC: (toluene/acetone/MeOH 6/1/1/ v/v): R_f = 0.39. m.p.: 136.4-137.3 °C. UV/Vis (CH₂C1₂): _max = 454 nm. ^-NMR: 4.294 (t, 2H, H-l), 2.985 (t, 2H, H- 2), 2.756 (t, 4H, Η-4/Η-Γ), 3.625 (t, 4H, H-5/H-2^'). ¹³C-NMR: 62.434 (CH₂, C- 1), 53.958 (CH₂, C-2), 56.766 (CH₂, C-4/C-1 , 59.819 (CH₂, C-5/C2^'). HRMS (ESI): C36H53NO4 calcd. 563.3975 (M⁺), found 563.3997.

63 2-(N,N-di(2-hydroxyethyl)-amino)ethyl-fi-apo-8 '-carotenoate hydrobromide (4) The diethanolamine analogue 11 (586 mg, 1.04 mmol) was dissolved in MeOH (30 mL) and HBr (0.1 M in water, 20.8 mL, 2.08 mmol) was added. The mixture was stirred at room temperature under N₂ for 3 h. The solvent was removed under reduced pressure and water by freeze-drying. The residue was dissolved in CH2CI2 and crystallized by addition of hexane giving 4 (587 mg, 90%). m.p.: 195.4-199.5 °C. UV/Vis (CH₂C1₂): λ_ΜΧ = 458 ran. ^-NMR: 4.675 (b, 2H, H-l), 3.775 (b, 2H, H-2), 3.543-3.656 (s, 4H, Η-4/Η-Γ), 4.074-4.157(b, 4H, H-5/H- 20, 9.301 (b, 1H, NH). ¹³C-NMR: 58.080 (CH₂, C-l), 55.375 (CH₂, C-2), 56.085 (CH₂, C-4/C-1 , 55.701 (CH₂, C-5/C-2^'), 53.142 (CH₃, C-17C-1 ^"). HRMS (ESI): C36H54NO4 calcd. 564.4052 (M⁺), found 564.4053.

2-(N,N-di(2-hydroxyethel), N-memyl-ammonium)ethyl-fi-apo-8 '-carotenoate iodide (5)

The diethanolamine analogue 11 (309.4 mg, 0.55 mmol) was dissolved in dry THF (50 mL) and Mel (3 mL) was added. The mixture was stirred at room temperature under N2 for 24 h and the crude product was separated by filtration. The quaternized ammonium cation 5 was recrystallized from CH2CI2 and hexane (308 mg, 80%). TLC (CHCl₃/MeOH/H₂0, 70/30/3 v/v): R_f = 0.33. m.p.: 199.8- 203.1 °C. UV/Vis (CH₂C1₂): λ_ΜΧ = 464 nm. ^-NMR: 4.678 (b, 2H, H-l), 4.128 (b, 2H, H-2), 3.924 (s, 4H, Η-4/Η-Γ), 4.213 (b, 4H, H-5/H-2^'), 3.474 (s, 3H, H- \ ~). ¹³C-NMR: 58.192 (CH₂, C-l), 62.882 (CH₂, C-2), 65.076 (CH₂, C-4/C-1 , 55.861 (CH₂, C-5/C-2 , 51.476 (CH₃, C-l ^"). HRMS (ESI): C₃7H₅₆N0₄ calcd. 578.4209 (M⁺), found 578.4208.

Biological methods

Synthetic cationic lipids C30-12, C30-14, C30-16, C30-18 and C30-20, as well as the commercial cationic lipid EPC, were formulated with either DOPE or cholesterol into liposomes. Liposomes were further formulated into lipoplexes by combination with plasmid DNA. Neutral cholesterol or neutral, zwitterionic DOPE were not included in the final calculation of charge ratios when forming lipoplexes between cationic lipids and DNA.

All lipids were initially dissolved in CH2CI2 in round bottom flasks, followed by evaporation of the organic solvent under reduced pressure resulting in thin films. Ethanol was then added to achieve stock solutions of 1 mM, Care was taken to protect the lipids from air and ambient light. All stock solutions were stored in amber vials under an inert atmosphere (N₂ or Ar) and kept at -80 °C.

An overall 3:2 molar ratio of total cationic lipid to co-lipid, either DOPE or cholesterol, in ethanolic solutions were prepared separately and evaporated under reduced pressure to generate thin films. The lipid films were hydrated with a known amount of sterile water to give 2 mM final hydrated stock solutions, which were stored overnight at 4 °C. Before use, the hydrated stocks were warmed to 37 °C and sonicated for 30 min.

Lipoplexes of concentrations 0.081 mM, 0.243 mM, 0.486 mM, 0.81 mM and 1.62 mM, corresponding to the N/P (+/-) molar charge ratios of 0.5 :1, 1.5: 1, 3:1 , 5.0: 1 and 10.0:1 , respectively, were prepared from the 2 mM liposome stocks. OPTI-MEM cell culture medium (57.6 μΕ) and DNA in E- Toxate™ Water, (14.4 μΕ; 250 ng/μΕ) were first combined, followed by the addition of an equal volume of corresponding liposome (72 μΕ) to this and mixed. These lipoplex formulations were incubated at 22 °C for 30 min.

For Lipid/siRNA Lipoplexes, lipid/siRNA lipoplexes were formulated by adding 54 μΕ of OPTI-MEM^® (Gibco Cell culture, CA) with 6 μΕ of siRNA (either GL2 or control) to give siRNA aliquots. Liposomes were diluted in OPTI-MEM^® to get 60 μΐ, Aliquots of desired molar concentration. SiRNA aliquots were added to the microcentifuge tubes containing the diluted liposomes, mixtures were pipette thoroughly and incubated for 20 min at room temperature before adding 180 μL· of OPTI-MEM^® to each formulation and applying them on the cells as described in the assay section.

The hydrodynamic diameters, du, of liposomes and lipoplexes were measured by dynamic light scattering (DLS, Malvern Zetasizer APS, Malvern, Worcestershire, UK) at 25 °C with a detection angle of 90°. All data are the mean + standard deviation (SD) of three measurements.

To 20 μL· of the lipoplexes, 2 μL· of the gel loading dye (6X) was added and mixed by pipetting. Eighteen microliters of each sample was then loaded onto a 1% agarose gel impregnated with ethidium bromide and run at 105 V for 1 h in lx TBE buffer. The migration of DNA complexed with the cationic lipids was impeded in the electric field. The DNA bands were observed using an ultraviolet transilluminator.

65 Twenty microliters of the lipoplexes was incubated with DNase I (2 units) at 37 °C for 1 h. After incubation, 5% SDS (4 μί) was added and incubated for a further 30 min, followed by 2 μL· of gel loading dye (6x).

Eighteen microliters of each lipoplex sample was then loaded onto a 1 % agarose gel impregnated with ethidium bromide and run at 105 V for 1 h in lx TBE buffer. The pDNA bands were observed using an ultraviolet transilluminator.

CHO-K1 cells were grown in RPMI media supplemented with 10% fetal calf serum and 100 U/mL of penicillin/streptomycin and 0.25 μg/mL amphotericin B. Cells were seeded 48 h before transfection onto opaque and transparent 96-well plate at a density of 10⁴ cells per well and incubated at 37 °C in presence of 5% C0₂ atmosphere. Cells were grown to 80% confluence before being washed with lx PBS and incubated with lipoplexes containing 3.6 μg of plasmid DNA in a volume of 45 μΕ in triplicate for 4 h at 37 °C in the presence of 5% C0₂ atmosphere. Complexes were then removed and the cells washed with lx PBS before adding 100 μΕ of complete RPMI media. Cells were left to incubate for an additional 44 h. Hydrated liposomal (cationic lipid / co- lipid) formulations (namely, EPC/Chol, C30-20/Chol and C20-20/Chol, all at a cationic lipid/co-lipid molar ratio of 3 :2) were generated from stock solutions from thin films by combining the required amounts of each alcohol solution of lipid and co-lipid, as determined by calculation of desired ratios, and removing the ethanol under reduced pressure. The thin films were then dissolved in a known amount of sterile water, followed by sonication to give a 2mM final solution of hydrated stocks. These hydrated stock liposomal solutions were stored overnight at 4°C. Before use, the hydrated stocks were warmed to 37 °C for 5 minutes in a water bath, then sonicated for 30 minutes.

PMOs are unable to enter cells in vitro due to their lack of charge.

Charge is introduced by annealing the PMOs to complementary

phosphorothioate-capped oligodeoxynucleotide leashes. The complementary sequence of the PMO is 17 bases long, with tails at either end. The tails of the leash are always of the sequence 'gattg' (5' to 3 ') at the 5' end of the PMO, and

'gtgat' (5 ' to 3 ') at the 3' end of the PMO. Leash/PMO stocks were prepared at

100 μΜ by mixing 12.5 μΐ ΙΟχ PBS with 7.5 μΐ RNase-, DNase-free H₂0, 25 μΐ leash (200 μΜ) and 5 μΐ PMO (1 mM) and annealed by gradual decrease in temperature from 94 °C in a thermal cycler, according to the method

66 recommended by Gebski, B.L.; Mann, C.J. ; Fletcher, S.; Wilton, S.D.

Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 2003, 12, 1801-1811.

Leashed PMOs were stored at 4°C for a maximum of 6 weeks.

Verification of annealing PMO to leash was established by running aliquots of leash alone and leashed PMO on a 3% agarose gel; an increase in size should be evident in leashed PMO relative to PMO alone, if PMO and leash have hybridized effectively.

Lipid/PMO-AO lipoplexes were formulated by adding equal volumes of liposome solution to PMO- AO at the desired charge ratio. The liposome particles were serially diluted to obtain varying cationic lipid / leash (N/P, or +/-) molar charge ratios at a given volume.

A gel retardation assay is a common technique, used in the context of this proposal to study the interaction between cationic lipids and AO. Briefly, the lipid/PMO-AO complexes, incubated in 20 mM HEPES, pH 5.5 were mixed with loading dye (bromophenol blue) and loaded onto the 3% agarose gel impregnated with ethidium bromide. The gel was then run at 105 V for 120 minutes in TBE buffer. The rate at which different molecules move through the gel was determined by their size and charge, and to a lesser extent, their shape.

Cells were seeded in a 6-well culture plate at a density of 8xl0⁴ cells/well and cultured till -80% confluence. The growth medium was removed and replaced with 2 ml pre-warmed differentiation medium and cells incubated at 37 °C, 5% C0₂ for 1 hour. During this 1-hour incubation with differentiation medium, the lipid:leashed PMO:DMEM mixes were prepared and incubated for 30 mins at RT. The differentiation medium was removed and wells were rinsed with 2 ml DMEM. A 1 ml aliquot of lipid:PMO:DMEM mix was added into each well and cells were incubated at 37 °C, 5% CO₂ for 4 hours. The lipid:PMO:DMEM mix was then replaced with 2 ml pre-warmed differentiation media. RNA was extracted after 24 hours.

RNA extraction and purification was performed using the QIAgen RNeasy mini kit. In brief, cells were lysed with buffer RLT, and lysates were homogenized with a QIAshredder column. RNA was purified with a RNeasy mini column containing an silica-gel membrane, washed with RW1 and RPE

67 buffers and eluted with RNase-free H₂0 and the concentration were measured using a ND-1000 Spectrophotometer (©Nano Drop).

Cells grown in an opaque 96-well plate were evaluated for β- galactosidase activity 48 hr after transfection using a Beta-Glo^® Assay System (Promega) according to the manufacturer's instructions. Luminescence was determined on a Perkin Elmer Precisely Wallac Envision 2104 Multilabel Plate reader (Perkin Elmer, Waltham, MA). β-Galactosidase activity was expressed as relative light units produced by the luminescence of luciferin, which was normalized for protein content. Total protein content was measured using Pierce^® BCA Protein Assay (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. A calibration curve obtained from a bovine serum albumin standard solution was used to determine cellular protein content per well.

The cytotoxicity associated with the lipoplex formulations at N:P (+/-) molar charge ratios ranging from 0.5:1 to 10:1 was evaluated using the MTS (3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) assay. Forty-eight hours after the application of lipoplexes, CHO- Kl cells in the transparent 96-well plates were washed with lx PBS, 50 μΕ of DMEM (phenol red-free media) and evaluated for cytotoxicity using the CellTiter96^® Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's instructions. The absorbance of converted dye, which correlates with the number of viable cells, was measured at 492 nm using a Victor Envision high throughput plate reader. The percentage of viable cells was calculated as the absorbance ratio of treated to untreated cells.

Structural information, such as the nature of the lipoplex packing morphologies and bilayer-bilayer distance, was obtained by SAXS (Small-angle X-ray scattering) analysis. Owing to the ordered packing a diffraction pattern is superimposed on the SAXS-curve. The SAXS experiments were performed at the European Synchrotron Radiation Facility (ESRF) on the bending magnet, BM29 BioSAXS beam line. BM29 is equipped with a double multilayer monochromator (energy band pass ~ 10^"2) and 4 mrad torodial mirror 1.1 m long. The experimental hutch is equipped with a marble table housing the modular- length flight tube, 2D detector (Pilatus 1M) and a sample handling equipment (automated sample changer). The sample-to-detector distance was 2.8 m.

68 Samples were prepared in 1 mL Eppendorf tubes, and loaded into the 1.8 mm diameter quartz capillary sample cell by an automated sample changer. Data collection was performed in 16-bunch mode at an energy of 12.5 keV with an exposure time of 2 s per frame using the dedicated beam line software

BsxCuBE. The SAXS-curves were obtained by integration and averaging of 20 2D images. Subsequent processing (such as background subtraction and scaling) and analysis were performed using the ATS AS -software package [Ref: Konarev, P. V.; Volkov, V. V.; Sokolova, A. V.; Koch, M. H. J.; Svergun, D. I., PRIMUS - a Windows-PC based system for small-angle scattering data analysis. /. Appl. Cryst. 2003, 36, 1277-1282.] and MATHEMATICA [Ref: Mathematica Version 8.0 Wolfram Research Inc. : Champaign, IL; University of Illinois Press, 2010.]. Cell culture. Cationic lipid mediated transfection of siRNA duplex (GL2) for specific knockdown of the luciferase transcript as well as of validated control siRNA, was performed using HR5-CL11 cells following standard methods. Briefly, HR5-CL11 cells were grown in DMEM media supplemented with 10% fetal calf serum and 100 U/mL of penicillin/streptomycin and the equivalent of 1 μg/ml doxycycline. Cells were seeded 24 h before transfection onto opaque and transparent 96-well plate at a density of 10⁴ cells per well and incubated with a 5% C0₂ atmosphere at 37 °C. Cells were grown to 80% confluence before being washed with PBS and incubated with 50 μΕ of each lipid-siRNA complex in triplicate for 4 h at 37 °C. The complexes were then removed by aspiration and the cells washed with PBS before adding 100 μΕ of DMEM media containing 2 μg/mL of doxycycline to each well. Cells were left to incubate additional 44 h. Following the incubation cells were used for the assays of luciferase, total proteins and cytotoxicity following the bellow mentioned protocols.

Luciferase knockdown assay. Forty-eight hours after the application of lipoplexes, treated cells in the opaque 96-well plates were washed with PBS, and lysed by adding 50 μΕ of Glo-Lysis™ buffer to each well. After a 15 min incubation period at room temperature, 50 μΕ of Bright Glo™ working solution, prepared according to the manufacturer's directions (Promega) were added to each well and mixed by pipetting. Luminescence was then read on a Victor Envision, high throughput plate reader.

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"Substantially" as the term is used herein means completely or almost completely; for example, a composition that is "substantially free" of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is "substantially pure" is there are only negligible traces of impurities present.

"Treating" or "treatment" within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder, or curing the disease or disorder.

Similarly, as used herein, an "effective amount" or a "therapeutically effective amount" of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition. In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects.

Phrases such as "under conditions suitable to provide" or "under conditions sufficient to yield" or the like, in the context of methods of synthesis,

72 as used herein refers to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within ordinary skill for an experimenter to vary, that provide a useful quantity or yield of a reaction product. It is not necessary that the desired reaction product be the only reaction product or that the starting materials be entirely consumed, provided the desired reaction product can be isolated or otherwise further used.

By "chemically feasible" is meant a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim. The structures disclosed herein, in all of their embodiments are intended to include only "chemically feasible" structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed or claimed herein.

An "analog" of a chemical structure, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure. A related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative."

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements will be apparent to those skilled in the art without departing from the spirit and scope of the claims.

All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by

73 preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

74

Claims

CLAIMS What is claimed is:

1. A carotenoid lipid of formula (I)

Car-C(=0)-L-Cat(+) X(-)

(I)

wherein Car is a carotenoid moiety, C(=0) is a carbonyl group bonded to a terminal ethenyl group of the carotenoid moiety, L is a linker bonded via an ester bond to the C(=0), linker, L optionally comprising a phosphate (Cl- C30)alkyl ester, Cat is a cationic quaternary ammonium, and X is an anion.

2. The carotenoid lipid of claim 1 wherein X is bromide or iodide.

3. The carotenoid lipid of formula (I) of claim 1 wherein Car is a moiety formu

or of formula (IIB)

wherein a wavy line indicates a point of bonding to the carbonyl group.

4. The carotenoid lipid of claim 1 wherein L is a group comprising a g phosphate ester group, the lipid being a carotenoid glycol phospholipid of formula

75 wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Q- C4)hydroxyalkyl, R⁴ is (Ci-C3o)alkyl, ml is 1, 2, or 3, m2 is 1, 2, or 3, and n is 1, 2, or 3. 5. The carotenoid glycol phospholipid (III) of claim 4 wherein each of R¹, R², and R³ is independently H, methyl, or hydroxyethyl.

6. The carotenoid glycol phospholipid of formula (III) of claim 4 wherein

wherein a wavy line indicates a point of bonding.

7. The carotenoid glycol phospholipid of formula (III) of claim 4 wherein n = 1.

8. The carotenoid glycol phospholipid of formula (III) of claim 4 wherein ml is 1.

9. The carotenoid glycol phospholipid of formula (III) of claim 4 wherein m2 is 1.

10. The carotenoid glycol phospholipid of formula (III) of claim 4 wherein R⁴ is C₂₀ alkyl.

11. The carotenoid glycol phospholipid of formula (III) of claim 4 wherein the compound is any one of

76

12. The carotenoid lipid of claim 1 wherein L is a chain comprising one, two, or three (C2-C4)oxyalkylene units bonded via an oxygen atom to the carbonyl group, the lipid being a carotenoid lycol lipid having formula (IV)

n2

77 (IV)

wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Q- C4)hydroxyalkyl, nl is 1, 2, or 3; and, n2 is 1, 2, or 3.

13. The carotenoid lipid of formula (IV) of claim 12 wherein each of R¹, and R³ is independently H, methyl, or hydroxyethyl.

14. The carotenoid lipid of formula (IV) of claim 12 wherein nl or both.

15. The carotenoid lipid of formula (IV) of claim 12 wherein the compound is any one of

78 wherein each of R¹, R², and R³ is independently H, (Ci-C4)alkyl, or (Q- C4)hydroxyalkyl, R⁴ and R⁵ are each independently selected (Ci-C3o)alkyl, and n is 1, 2, or 3; X is an anion.

17. The carotenoid glyceryl phospholipid of claim 16 wherein each of R¹, R², and R³ is independently H, methyl, or hydroxyethyl.

18. The carotenoid glyceryl phospholipid of claim 16 wherein Car is a

wherein a wavy line indicates a point of bonding.

19. The carotenoid glyceryl phospholipid of claim 16 wherein n = 1.

20. The carotenoid glyceryl phospholipid of claim 16 wherein R⁴ is a C₂o alkyl group.

21. The carotenoid glyceryl phospholipid of claim 16 wherein R⁵ is a C₂o alkyl group.

22. The carotenoid glyceryl phospholipid of claim 16 wherein the compound is any one of

79

24. The liposome of claim 23 comprising dioleoylphosphatidylethanolamine.

25. The liposome of claim 23 further comprising a polynucleotide contained therein.

26. The liposome of claim 25 wherein the polynucleotide comprises a si(RNA) or a DNA contained therein.

27. A method of transferring a polynucleotide to the interior of a living cell, comprising contacting the cell and an effective amount or concentration of the liposome of claim 25.

28. The method of claim 27 wherein the polynucleotide comprises a si(RNA) or a DNA.

80

29. The method of claim 27 wherein the living cell is within a living organism.

30. The method of claim 29 wherein an immune response is not induced by the contacting of the liposome and the cell within the living organism.

31. The method of claim 27 wherein the polynucleotide is incorporated into the genome of the cell, or interferes with expression of the genome of the cell, or both.

32. A method of monitoring the uptake of the liposome of claim 23 into a living cell, comprising observing an absorbance of visible light by the carotenoid moiety Car.

33. The method of claim 32 wherein observing is observing with the human eye.

34. The method of claim 32 wherein observing is observing with a spectrometer.

35. A method of treatment of a medical disorder in a patient wherein administration of a liposome containing a polynucleotide is medically indicated, comprising administering to the patient an effective amount of the liposome of claim 25.

36. The method of claim 35, wherein the medical disorder is Duchenne muscular dystrophy.

81