Multifunctional, stimuli-sensitive
`nanoparticulate systems for
`drug delivery
`
`Vladimir P. Torchilin1,2
`
`Abstract | The use of nanoparticulate pharmaceutical drug delivery systems (NDDSs)
`to enhance the in vivo effectiveness of drugs is now well established. The development
`of multifunctional and stimulus-sensitive NDDSs is an active area of current research.
`Such NDDSs can have long circulation times, target the site of the disease and enhance
`the intracellular delivery of a drug. This type of NDDS can also respond to local stimuli
`that are characteristic of the pathological site by, for example, releasing an entrapped
`drug or shedding a protective coating, thus facilitating the interaction between
`drug-loaded nanocarriers and target cells or tissues. In addition, imaging contrast
`moieties can be attached to these carriers to track their real-time biodistribution and
`accumulation in target cells or tissues. Here, I highlight recent developments with
`multifunctional and stimuli-sensitive NDDSs and their therapeutic potential for diseases
`including cancer, cardiovascular diseases and infectious diseases.
`
`Enhanced permeability
`and retention (EPR) effect
`The property through which
`macromolecules (such as
`nanoparticles) accumulate
`in areas of inflammation
`including tumours, owing to
`the increased vascular
`permeability or abnormal
`blood vessel architecture.
`
`1Center for Pharmaceutical
`Biotechnology and
`Nanomedicine, Northeastern
`University, 140 The Fenway,
`Room 214, 360 Huntington
`Avenue, Boston,
`Massachusetts 02115, USA.
`2Faculty of Pharmacy,
`King Abdulaziz University,
`Jeddah 21589, Saudi Arabia.
`e‑mail: v.torchilin@neu.edu
`doi:10.1038/nrd4333
`Published online
`7 October 2014
`
`Nanoparticulate pharmaceutical drug delivery systems
`(NDDSs) are widely used in pharmaceutical research
`and in clinical settings to enhance the effectiveness
`of diagnostic agents and drugs, including anticancer,
`antimicrobial and antiviral drugs1,2. The types of nano­
`carriers that exist are diverse and include the following:
`liposomes; polymeric nanoparticles; polymeric micelles;
`silica, gold, silver and other metal nanoparticles; carbon
`nanotubes; solid lipid nanoparticles; niosomes; and dendri­
`mers. The use of NDDSs can overcome several problems
`that are associated with traditional drugs, such as poor
`aqueous solubility, low bioavailability and nonspecific
`distribution in the body.
`The first generation of NDDSs mainly aimed to
`address single challenges, such as the need to increase
`drug stability in vivo and the circulation time in the
`blood, or the need to target a drug to a specific tissue or
`pathology. Now, research has led to the development of
`NDDSs that can perform two or more functions (either
`simultaneously or sequentially) to overcome multiple
`physiological barriers to optimize delivery and deliver
`their loads (which can be single or multiple) to the
`required target sites (such as organs, tissues, cells) or
`specific pathologies in the body3 (FIG. 1). The properties
`of multifunctional NDDSs include the ability to bear
`a sufficient load of a drug or DNA­related material, have
`
`increased circulation times (through the use of soluble
`polymers) and target the intended site of action both
`nonspecifically (for example, via the enhanced permea­
`bility and retention (EPR) effect) and specifically (via the
`attachment of target­specific ligands). In addition,
`multifunctional NDDSs can respond to several stimuli
`that are characteristic of the pathological site, which
`is achieved through the inclusion of components that
`react to abnormal pH, temperature and redox condi­
`tions, and to the overexpression of certain biological
`molecules. Multifunctional NDDSs can also respond
`to stimuli from outside the body, such as magnetic or
`ultrasound fields, and can be supplemented with an
`imaging contrast moiety to enable their biodistribution,
`target accumulation or the efficacy of the therapy to be
`monitored.
`Although as yet there is no broadly recognized and
`accepted single classification system for multifunctional
`NDDSs, they can generally be divided into three groups.
`The first group consists of drug­loaded NDDSs that com­
`bine at least two different functions, such as longevity,
`targetability, stimuli­sensitivity or cell penetration. The
`second group of NDDSs, in addition to the previously
`described properties, are loaded with more than one drug
`and/or gene therapy­related material, such as antisense
`oligonucleotides or small interfering RNAs (siRNAs).
`
`NATURE REVIEWS | DRUG DISCOVERY
`
` VOLUME 13 | NOVEMBER 2014 | 813
`
`© 2014 Macmillan Publishers Limited. All rights reserved
`
`REVIEWS
`
`

`

`Multifunctional, stimuli-sensitive
`nanoparticulate systems for
`drug delivery
`
`Vladimir P. Torchilin1,2
`
`Abstract | The use of nanoparticulate pharmaceutical drug delivery systems (NDDSs)
`to enhance the in vivo effectiveness of drugs is now well established. The development
`of multifunctional and stimulus-sensitive NDDSs is an active area of current research.
`Such NDDSs can have long circulation times, target the site of the disease and enhance
`the intracellular delivery of a drug. This type of NDDS can also respond to local stimuli
`that are characteristic of the pathological site by, for example, releasing an entrapped
`drug or shedding a protective coating, thus facilitating the interaction between
`drug-loaded nanocarriers and target cells or tissues. In addition, imaging contrast
`moieties can be attached to these carriers to track their real-time biodistribution and
`accumulation in target cells or tissues. Here, I highlight recent developments with
`multifunctional and stimuli-sensitive NDDSs and their therapeutic potential for diseases
`including cancer, cardiovascular diseases and infectious diseases.
`
`Enhanced permeability
`and retention (EPR) effect
`The property through which
`macromolecules (such as
`nanoparticles) accumulate
`in areas of inflammation
`including tumours, owing to
`the increased vascular
`permeability or abnormal
`blood vessel architecture.
`
`1Center for Pharmaceutical
`Biotechnology and
`Nanomedicine, Northeastern
`University, 140 The Fenway,
`Room 214, 360 Huntington
`Avenue, Boston,
`Massachusetts 02115, USA.
`2Faculty of Pharmacy,
`King Abdulaziz University,
`Jeddah 21589, Saudi Arabia.
`e‑mail: v.torchilin@neu.edu
`doi:10.1038/nrd4333
`Published online
`7 October 2014
`
`Nanoparticulate pharmaceutical drug delivery systems
`(NDDSs) are widely used in pharmaceutical research
`and in clinical settings to enhance the effectiveness
`of diagnostic agents and drugs, including anticancer,
`antimicrobial and antiviral drugs1,2. The types of nano­
`carriers that exist are diverse and include the following:
`liposomes; polymeric nanoparticles; polymeric micelles;
`silica, gold, silver and other metal nanoparticles; carbon
`nanotubes; solid lipid nanoparticles; niosomes; and dendri­
`mers. The use of NDDSs can overcome several problems
`that are associated with traditional drugs, such as poor
`aqueous solubility, low bioavailability and nonspecific
`distribution in the body.
`The first generation of NDDSs mainly aimed to
`address single challenges, such as the need to increase
`drug stability in vivo and the circulation time in the
`blood, or the need to target a drug to a specific tissue or
`pathology. Now, research has led to the development of
`NDDSs that can perform two or more functions (either
`simultaneously or sequentially) to overcome multiple
`physiological barriers to optimize delivery and deliver
`their loads (which can be single or multiple) to the
`required target sites (such as organs, tissues, cells) or
`specific pathologies in the body3 (FIG. 1). The properties
`of multifunctional NDDSs include the ability to bear
`a sufficient load of a drug or DNA­related material, have
`
`increased circulation times (through the use of soluble
`polymers) and target the intended site of action both
`nonspecifically (for example, via the enhanced permea­
`bility and retention (EPR) effect) and specifically (via the
`attachment of target­specific ligands). In addition,
`multifunctional NDDSs can respond to several stimuli
`that are characteristic of the pathological site, which
`is achieved through the inclusion of components that
`react to abnormal pH, temperature and redox condi­
`tions, and to the overexpression of certain biological
`molecules. Multifunctional NDDSs can also respond
`to stimuli from outside the body, such as magnetic or
`ultrasound fields, and can be supplemented with an
`imaging contrast moiety to enable their biodistribution,
`target accumulation or the efficacy of the therapy to be
`monitored.
`Although as yet there is no broadly recognized and
`accepted single classification system for multifunctional
`NDDSs, they can generally be divided into three groups.
`The first group consists of drug­loaded NDDSs that com­
`bine at least two different functions, such as longevity,
`targetability, stimuli­sensitivity or cell penetration. The
`second group of NDDSs, in addition to the previously
`described properties, are loaded with more than one drug
`and/or gene therapy­related material, such as antisense
`oligonucleotides or small interfering RNAs (siRNAs).
`
`NATURE REVIEWS | DRUG DISCOVERY
`
` VOLUME 13 | NOVEMBER 2014 | 813
`
`© 2014 Macmillan Publishers Limited. All rights reserved
`
`REVIEWS
`
`

`

`Liposome, micelle
`Drug A
`Drug B
`
`Moiety sensitive to:
`• pH
`• Temperature
`• Redox
`• Enzyme activity
`
`Polymer coat, such
`as PEG, to increase
`longevity
`
`Targeting agent:
`• Antibodies
`• Transferrin
`• Peptides
`
`Cell-penetrating
`peptide such as
`HIV TAT peptide
`
`Imaging or
`contrast agent:
`• Gd
`• 64Cu
`
`Figure 1 | Schematic of a drug-loaded, multifunctional, stimuli-sensitive NDDS.
`Drugs (Drug A and Drug B) can be loaded into a pharmaceutical nanocarrier, such as
`Nature Reviews | Drug Discovery
`a liposome or polymeric micelle. Depending on the purpose of the nanoparticulate
`pharmaceutical drug delivery system (NDDS), various agents can be added to the
`nanoparticle to target the NDDS to a particular tissue, to increase cell penetration,
`to enable imaging or to release the drugs in response to a given stimulus.
`PEG, poly(ethylene glycol).
`
`The third group consists of so­called theranostic NDDSs,
`which have an additional diagnostic label for use with
`current clinical imaging modalities.
`Research in the area of multifunctional NDDSs4,5 is
`very active, but substantial work remains to make them
`a clinical reality. Here, I highlight recent developments
`relating to multifunctional NDDSs. The majority of the
`currently available data relate to cancer, although there
`are some examples with other diseases.
`
`NDDS longevity and targeting
`One of the most common uses of NDDSs is to com­
`bine prolonged circulation times with targetabilty. Such
`NDDSs are particularly useful for tumour targeting
`because tumours (as well as other inflammation zones)
`usually have increased vascular permeability as well as
`poor lymphatic drainage6,7. This enables long­circulating
`NDDSs to accumulate in tumours through the EPR
`effect, which forms the basis for passive targeting8.
`Never the less, EPR­based drug delivery strategies face
`several challenges. First, tumours — especially large,
`solid tumours — are pathophysiologically heterogene­
`ous. Some parts of such tumours are not vascularized,
`do not exhibit the EPR effect, may have sizeable necrotic
`areas9,10 and have varied microvascular permeability10.
`In addition, the increased interstitial pressure that exists
`within tumours may limit the EPR­mediated accumula­
`tion of NDDSs even if the vasculature is leaky11.
`NDDSs that are used for passive targeting and/or
`spontaneous accumulation must have long circulation
`times to ensure that sufficient drug is delivered to the tar­
`get tissue. The usual approach to obtain long­circulating
`NDDSs is to coat them with hydrophilic and flexible
`polymers, such as poly(ethylene glycol) (PEG), as was
`first suggested for liposomes12. The pegylation of NDDSs
`prevents their interaction with opsonins and impedes
`their capture and clearance by the mononuclear phago­
`cyte system. However, pegylated NDDSs can induce the
`production of antibodies that can accelerate the blood
`clearance of nanoparticles (as was demonstrated for
`
`Passive targeting
`The mechanism through
`which nanoparticulate
`pharmaceutical drug delivery
`systems tend to accumulate
`in tumours, probably through
`the enhanced permeability
`and retention effect.
`
`Quantum dots
`Nanometre­scale particles of
`semiconductor materials that
`have quantum mechanical
`properties.
`
`Active targeting
`The mechanism through which
`specific moieties attached to
`nanoparticulate pharmaceutical
`drug delivery systems force
`them to interact with a specific
`type of cell or tissue.
`
`clinically used pegylated liposomes), particularly after
`repetitive administration; this is the so­called accelerated
`blood clearance phenomenon13,14.
`Although PEG is still the gold­standard polymer
`that is used to prepare long­circulating NDDSs, other
`hydrophilic polymers that are used include poly[N­
`(2­hydroxypropyl) methacrylamide]15, poly(acryloyl
`morpholine), poly­N­vinylpyrrolidones16 and polyvinyl
`alcohol17. In general, the concept of pegylation, as well as
`the use of alternative polymers for longevity, is well
`established and reviewed; thus, in the next section only
`some of the key developments in this area are briefly
`mentioned. Importantly, the shape of NDDSs can also
`influence their pharmacokinetics and biodistribution18,19.
`Liposomal long­circulating NDDSs are the most
`frequently studied type of NDDS; however, synthetic
`amphiphilic polymers have also been used to sterically
`stabilize several other types of NDDS to alter their bio­
`distribution. For example, pegylation of gold nanoparti­
`cles reduced their uptake by the mono nuclear phagocyte
`system and their subsequent clearance from the body20.
`One potential application of pegylated gold nanoparticles
`is their use in photothermal tumour therapy (known as
`ablation) following their accumulation in the tumour21.
`Pegylated methotrexate­conjugated poly­l­lysine
`dendrimers also accumulated efficiently in solid tumours
`in rats and mice via the EPR affect22, and pegylation
`reduced the toxicity of positive charge­bearing dendri­
`mers in cell­culture experiments23. Quantum dots can also
`be modified by pegylation. In mice, pegylated quantum
`dots had prolonged circulation times, which were attrib­
`utable to their decreased uptake in organs (such as the
`spleen and liver) in which the mononuclear phagocyte
`system is active24.
`Active targeting of NDDSs can be achieved by attaching
`targeting ligands, such as monoclonal antibodies, trans­
`ferrin, various peptides, folate, aptamers (single­stranded
`oligonucleotides) or certain sugar moieties, onto their
`surface25,26. To prevent steric hindrance between the tar­
`geting moiety and the protective polymer (such as PEG),
`the targeting ligand is usually attached to the chemically
`activated distal end of the NDDS­grafted polymeric
`chain27. An example of such active targeting of NDDSs
`is pegylated doxorubicin­loaded liposomes that have
`human epidermal receptor 2 (HER2; also known as
`ERBB2)­specific antibodies attached; these were suc­
`cessfully used to target HER2­overexpressing SK­BR3
`cells in mice28. A nucleosome­specific monoclonal
`antibody (mAb 2C5) that recognized multiple types of
`tumour cells via tumour cell­surface­bound nucleosomes
`improved the targeting of doxorubicin­loaded liposomes
`to tumour cells and increased cancer cell cytotoxicity in
`in vitro and in vivo models29, including nude mice xeno­
`grafted with the U­87 cell line, which was derived from
`a human glioblastoma30. In another example of active
`targeting, pegylated gold nanoparticles conjugated to
`monoclonal F19 antibodies were used as targeted label­
`ling agents for human pancreatic carcinoma tissues31.
`Lactoferrin­conjugated PEG–polylactic­acid nanoparti­
`cles improved the delivery of the conjugated particles to
`the brain in an experimental murine model32.
`
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`
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`
`R E V I E W S
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`

`An interesting approach to increase targeting centres
`on self­assembling polyalkylcyanoacrylate­based nano­
`particles. These types of nanoparticles could serve as
`pharmaceutical carriers for various drugs and could be
`produced in pegylated and ‘activated’ forms, enabling
`various ligands to be easily attached33. Polyisohexyl­
`cyanoacrylate nanoparticles loaded with doxorubicin
`produced by BioAlliance Pharma as Livatag (doxoru­
`bicin Transdrug) are currently in a Phase III trial in
`Europe and in the United States as a second­line treat­
`ment of hepatocellular carcinoma after sorafenib, at a
`stage with no available approved treatment (Livatag
`press release; see Further information).
`Aptamers that impart affinity and specificity by elec­
`trostatic, hydrogen or hydrophobic bonding, but not via
`the base pairing, have also been used as stable and effi­
`cient targeting moieties for NDDSs. For example, lipo­
`somal NDDSs modified with specific aptamers have
`been used to target leukaemia cells34.
`Targetability can also be applied to stimuli­sensitive
`NDDSs. Gemcitabine­loaded pegylated pH­sensitive
`liposomes modified with an epidermal growth factor
`receptor (EGFR)­specific antibody efficiently inhib­
`ited tumour growth in mice35. Micellar NDDSs that
`were composed of amphiphilic conjugates of PEG and a
`co­polymer of poly(ε­caprolactone) and poly(malic acid)
`loaded with doxorubicin conjugated to poly(malic acid)
`via a pH­sensitive bond were modified with folate for
`enhanced cellular uptake36. This preparation provided an
`enhanced release of doxorubicin at lowered pH values
`inside cancer cells36. NDDSs with similar properties
`based on pH­sensitive polymethacrylate (PMA)­grafted
`poly(amidoamine) nanocarriers and additionally
`pegylated and modified with a folate moiety efficiently
`delivered paclitaxel to tumours and inhibited tumour
`growth in mice37.
`Clearly, the separation of long­circulating NDDSs into
`those that achieve active targeting and passive targeting is
`conditional. That is, both phenomena are closely connected
`because NDDSs will accumulate in the target area via the
`EPR effect (passive targeting) before ligand­mediated
`interaction with target cells (active targeting) occurs.
`Another interesting issue is associated with the use of
`targeting ligands that are also naturally present (in their
`free form) in the circulation, such as folate, transferrin or
`certain peptides. Although one may expect competition
`between the ligands attached to NDDSs and the native
`ligands for the binding sites, the success of folate­ and
`transferrin­targeted NDDSs (see below) demonstrates
`that this potential problem can be overcome. This is
`probably achieved through multipoint interactions of
`ligand­modified NDDSs with the target and/or because
`of the rapid recirculation of the cognate receptors, which
`provides sufficient opportunities for an interaction of the
`NDDS with the receptor.
`
`Disease applications for NDDS-based therapies
`Cardiovascular diseases. NDDSs hold promise as thera­
`peutic, diagnostic and theranostic agents for cardio­
`vascular diseases38, particularly atherosclerosis39 (FIG. 2).
`Multifunctional micellar NDDSs that combined a
`
`targeting pentapeptide, a fluorophore and a drug targeting
`atherosclerotic plaques by specifically binding to clotted
`plasma proteins were used to visualize atherosclerotic
`lesions in a mouse model. This preparation was shown
`to increase the amount of the anticoagulant agent biva­
`lirudin that was delivered to the lesion40. Because the
`increased uptake of low­density lipoproteins (LDLs)
`by resident macrophages in plaques is associated with
`the progression of atherosclerosis, targeting plaque­
`associated macrophages could be a method of inhibit­
`ing LDL uptake41. Indeed, targeted polymeric NDDSs
`loaded with pravastatin specifically and dramatically
`inhibited the phagocytic activity of macrophages without
`affecting non­target cells42.
`Multimodal diagnostic micellar NDDSs that were
`fluorescent and paramagnetic and targeted to macro­
`phage scavenger receptors using specific antibodies have
`been used to visualize lesions in the abdominal aortas
`of mice with atherosclerosis43. Furthermore, polymeric
`NDDSs made of polyketals that were used to deliver
`small­molecule drugs, such as the p38 mitogen­activated
`protein kinase (MAPK) inhibitor SB239063, and anti­
`oxidant proteins, such as superoxide dismutase, to the area
`of the myocardial infarction in a rat model significantly
`improved cardiac function44.
`Liposome­based systems have also been frequently
`used to study the potential use of NDDSs in cardiovascular
`diseases45. For example, liposomes loaded with ATP or
`co­enzyme Q accumulated well in the infarcted areas of
`the myocardium and improved cardiac parameters in
`rat and rabbit models of myocardial infarct46,47. In a rat
`model of heart transplant, organ rejection was simulta­
`neously imaged and treated using multifunctional poly­
`meric NDDSs co­loaded with superparamagnetic iron
`oxide nanoparticles (SPIONs) and plasmid DNA48. The
`SPION acted as a magnetic resonance imaging (MRI)
`contrast agent, whereas the plasmid DNA suppressed the
`local immune response48. NDDSs for targeted thromb­
`o lytic therapy were developed by conjugating a throm­
`bolytic agent (recombinant tissue plasminogen activator)
`to dextran­coated iron oxide nanoparticles, which were
`additionally modified with a thrombus­targeted pep­
`tide that was sensitive to activated factor XIII49. These
`NDDSs had increased binding to the margins of intravas­
`cular thrombi and good thrombolytic activity in a mouse
`model of pulmonary embolism.
`The translation of these NDDSs and others identified
`in similar studies into clinical practice remains the primary
`challenge to advancing the field and will be addressed in
`the closing section of this article.
`
`Infectious diseases. NDDSs have a growing role in com­
`batting infectious diseases50. Of particular interest here
`are multicomponent NDDSs that contain metals, which
`can facilitate the formation of reactive oxygen species
`to eliminate pathogenic bacteria (FIG. 2). For example,
`NDDSs that combined iodinated chitosan and silver
`nanoparticles bound to and killed bacteria better than
`either component alone51. Hydrogel and glass­based
`nanoparticles capable of releasing nitric oxide facili­
`tated the cleaning and healing of open wounds in mice52.
`
`NATURE REVIEWS | DRUG DISCOVERY
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` VOLUME 13 | NOVEMBER 2014 | 815
`
`© 2014 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
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`

`a Cardiovascular disease
`
`Atherosclerotic
`plaque
`
`Heart
`
`b Infectious disease
`
`Clot
`proteins
`
`NDDS
`
`Macrophage
`
`Clot
`proteins
`
`ROS
`
`Protein synthesis
`
`Bacterium
`
`Metals
`
`Antibiotics
`
`Figure 2 | Multifunctional and stimuli-sensitive NDDSs in cardiovascular pathologies and infectious diseases.
`Nature Reviews | Drug Discovery
`a | Nanoparticulate pharmaceutical drug delivery systems (NDDSs) can be engineered to target activated macrophages
`in atherosclerotic plaques or proteins present in a clot. These nanoparticles can thereby reduce plaque size through
`these two distinct mechanisms. b | NDDSs can be used to deliver metals to bacteria, where they can generate reactive
`oxygen species (ROS) to induce membrane blebbing and DNA damage. Antibiotics can also be delivered intracellularly
`using NDDSs.
`
`NDDSs can be used to deliver antibiotics inside cells;
`murine salmonellosis was successfully treated (with
`a greater than tenfold decrease in the level of pathogen
`in the liver and spleen of experimental mice) using gen­
`tamicin in silica xerogel­based NDDSs53. The bacteria­
`mediated degradation of a polyphosphoester core of
`antibiotic­loaded NDDSs by bacterial enzymes54 pro­
`vided an interesting example of a targeted, responsive
`NDDS. Nevertheless, this area is in early stages of devel­
`opment and more animal­based studies will be required
`to reveal its full potential.
`
`Cancer. The majority of preclinical research with NDDSs
`relates to cancer. Based on one of the most popular
`pharmaceutical nanocarriers — liposomes — several
`anticancer NDDSs are available at different stages of
`development (see TABLE 1 for a selected list).
`The next step in the development of anticancer NDDSs
`is to target them to tumours. Cancer cells contain sev­
`eral targets discussed below that can be used by NDDSs.
`Each of these targets could be specific for a certain type
`or types of cancer. Although it is possible that there will
`be overlapping expression of a target in cancer cells and
`normal cells, the level of expression is usually much higher
`in cancer cells; this characteristic is a prerequisite for an
`NDDS­targeting ligand.
`Folate has been used as a targeting ligand in pre­
`clinical studies of NDDSs, owing to its easy conjugation
`to nanocarriers, high affinity for folate receptors and the
`lower expression of folate receptors in normal cells then
`
`in cancer cells and activated macrophages that are typical
`of inflammatory diseases55. Numerous folate­ targeted
`liposomal systems for cancer have been described
`(reviewed in REFS 56,57). For example, liposomes modi­
`fied with folate via a PEG spacer efficiently delivered
`their cargo intracellularly through receptor­mediated
`endocytosis (which could bypass multidrug resistance),
`and doxorubicin­mediated cytotoxicity against cancer
`cells expressing folate receptors was 85­fold higher with
`drug­loaded targeted liposomes than with unmodified
`liposomes58. In addition, when glutathione­conjugated
`folate was coupled to a PEG–distearoylphosphatidyl­
`ethanolamine (PEG–DSPE) polymer and incorporated
`into the liposomal membrane, there was increased activity
`of the liposomal vincristine and favourable pharmaco­
`kinetics in mice59. Folate­based modified NDDSs loaded
`with paclitaxel60 or carboplatin61 had high antitumour
`activity in mouse models. Liposome targeting to folate
`receptors has also been used to improve gene delivery of
`the herpes simplex virus type 1 thymidine kinase suicide
`gene; the liposome preparation inhibited tumour growth
`in a mouse model of oral cancer62.
`Transferrin, an 80 kDa serum glycoprotein, binds
`to the transferrin receptor and is taken into cells via
`receptor­mediated endocytosis. Compared with normal
`cells, the expression of transferrin receptor in certain
`cancer cells is much higher because of their increased
`demand for iron. Consequently, transferrin­receptor­
`targeted NDDSs could be used for anticancer therapy.
`Indeed, this was demonstrated with doxorubicin­loaded
`
`816 | NOVEMBER 2014 | VOLUME 13
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`
`© 2014 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
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`

`

`Table 1 | Liposome-based drugs for cancer therapy
`Drug formulation
`Indication
`Liposomal doxorubicin (Doxil, also known
`AIDS-related Kaposi’s sarcoma, recurrent
`as Caelyx; Janssen)
`ovarian cancer, metastatic breast cancer
`and multiple myeloma
`Metastatic breast cancer
`
`Status*
`Approved
`
`Approved
`
`Approved
`
`Phase III, active: NCT00617981
`Phase I/II, recruiting: NCT00826085
`
`Phase III, recruiting: NCT02112656
`Approved in China
`
`Phase II, completed: NCT01537536,
`NCT00448305
`Phase II, completed: NCT00377936
`Phase II, completed: NCT00542048
`Phase II, completed: NCT01190982
`
`Approved
`
`Approved
`
`Kaposi’s sarcoma, breast and ovarian
`cancer
`Hepatocellular carcinoma
`Breast cancer
`
`Hepatocellular carcinoma
`Solid tumours
`
`Breast cancer
`
`Pancreatic cancer
`Liver cancer
`Breast cancer
`
`Kaposi’s sarcoma
`
`Acute lymphoblastic leukaemia
`
`Non-pegylated liposomal doxorubicin
`(Myocet; Sopherion Therapeutics)
`Liposomal doxorubicin (LipoDox;
`Sun Pharma Global FZE)
`Thermally sensitive liposomal
`doxorubicin (Thermodox; Celsion )
`
`Liposomal paclitaxel (Lipusu; Luye
`Pharma)
`Liposomal paclitaxel (EndoTAG-1;
`Medigene)
`
`Liposomal paclitaxel (LEP-ETU;
`NeoPharm)
`Liposomal daunorubicin (DaunoXome;
`Galen)
`Liposomal vincristine (Marqibo; Talon
`Therapeutics)
`Liposomal cisplatin (LiPlaCis; LiPlasome
`Pharma)
`Liposomal cisplatin (Lipoplatin; Regulon)
`
`Liposomal cisplatin (SPI-077; Azla
`Corporation)
`Liposomal formulation of SN-38,
`the active metabolite of irinotecan
`(LE-SN38; NeoPharm)
`Liposomal irinotecan HCl: floxuridine
`mixture (CPX-1; Celator Pharmaceuticals)
`Liposomal irinotecan (MM-398;
`Merrimack Pharmaceuticals)
`Liposomal L-annamycin
`
`Refs
`165
`
`168,169
`
`170,171
`
`182–184
`
`166
`
`175,176
`
`202
`
`167
`
`172–174
`
`203
`
`175,
`177–179
`175,180,
`181
`175,185
`
`182
`
`204
`
`186,187
`
`188
`
`Advanced or refractory solid tumours
`
`Phase I, recruiting: NCT01861496
`
`Pancreatic cancer, non-small-cell lung cancer,
`head and neck cancer and breast cancers
`Ovarian cancer
`
`Phase I/II/III
`
`Phase II, completed: NCT00004083
`
`Colorectal carcinoma
`
`Phase II, completed: NCT00311610
`
`Colorectal cancer
`
`Phase II, completed: NCT00361842
`
`Metastatic pancreatic cancer
`
`Phase III, active: NCT01494506
`
`Acute lymphocytic leukaemia,
`doxorubicin-resistant blood cancer
`Hodgkin’s disease, non-Hodgkin’s lymphoma
`
`Phase I/II
`
`Phase I, active: NCT00364676
`
`Liposomal vinorelbine, INX-0125
`(AlocrestTM; Hana Biosciences)
`*ClinicalTrials.gov identifiers provided where appropriate.
`
`long­circulating transferrin­modified liposomes in a rat
`model of liver cancer63. Other examples of the anticancer
`effects of transferrin­targeted liposomes in mouse mod­
`els include those loaded with boron compounds64,65 and
`with ceramide66.
`EGFRs are frequently overexpressed in solid tum­
`ours67,68 and are therefore popular targets for NDDSs.
`HER2, which is overexpressed in approximately 20% of
`breast cancers68 and some other cancers, is also a popular
`target for NDDSs. For example, there was greater accu­
`mulation of HER2­targeted pegylated liposomes loaded
`with doxorubicin than of non­targeted liposomes in
`HER2­overexpressing breast cancer cells69 and in ascitic
`lymphoma cells70.
`
`The tumour vasculature is also an attractive target;
`destruction of the vasculature inhibits tumour growth
`and metastasis, it is not tumour type­specific and,
`importantly, NDDSs do not need to diffuse into the
`tumour mass to reach this target71. Targets that have
`been the focus of most studies include vascular endothe­
`lial growth factor (VEGF; especially for anti­angiogenic
`therapy), vascular cell adhesion molecule (VCAM),
`matrix metalloproteinases (MMPs) and integrins72,73.
`For example, peptides that bind specifically to the
`tumour vasculature coupled to liposomes loaded with
`doxorubicin improved drug efficacy against several
`human cancers in severe combined immunodeficient
`mice74. Pegylated VCAM­targeted immunoliposomes
`
`NATURE REVIEWS | DRUG DISCOVERY
`
` VOLUME 13 | NOVEMBER 2014 | 817
`
`© 2014 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
`
`

`

`bound selectively to activated endothelial cells in vitro
`and accumulated in tumour vessels in vivo75. MMPs are
`overexpressed on the newly formed vessels and tumour
`tissues, and are involved in tissue remodelling, tumour
`invasiveness, resistance to apoptosis and metastasis. An
`antigen­binding fragment of the antibody against MMP
`that was conjugated to doxorubicin­loaded liposomes
`via a PEG spacer showed enhanced tumoural uptake and
`inhibited tumour growth in a mouse model76.
`
`Considerations for NDDS receptor targeting. The success
`of receptor­mediated targeting depends strongly on the
`target affinity and the density of the receptors that are
`present on the cell surface. In addition, with pegylated
`NDDSs, the surface­attached PEG could prevent or hinder
`the interaction between the targeting ligand and its recep­
`tor. In a sense, this limits the opportunities of targeting
`because not all potential targets are expressed at sufficient
`levels. It is also important to consider, on a case by case
`basis, whether spontaneous targeting (passive accumula­
`tion; based on the EPR effect) or ligand­mediated specific
`targeting (which clearly requires more synthetic effort)
`is preferable. In general, if good accumulation of a drug
`in the pathological tissue is the primary goal, then EPR­
`mediated accumulation may suffice. However, if the pres­
`ence of the drug inside the cell is needed then ligands that
`can be internalized may be required.
`
`Stimuli-sensitive NDDSs
`The use of NDDSs that respond to different types of
`