in accordance with the goals of this special issue of metal - based drugs , this article deems to 
 fulfil a teaching and guiding role in new anticancer drug research .
we hope it will spawn new ideas and new 
 research in the field of anticancer therapy . in what follows , 

we first create an understanding of why new anticancer drugs are vigorously 
 searched for by highlighting some of the problems associated with a typical 
 chemotherapeutic agent , cisplatin .
one 
 possible way to minimise the negative side effects associated with 
 chemotherapeutic agents is to make use of a synthetic polymeric drug delivery 
 system .
we explain how such systems may 
 significantly enhance the therapeutic effectiveness of chemotherapeutic agents 
 and highlight some of the structural features they must have .
we then develop a general synthetic strategy 
 an experimental chemist may follow to actually synthesise a polymeric drug delivering 
 device and proceed to visualise this strategy utilising a literature 
 example .
the need for certain 
 structural features in a delivering device is then motivated by a discussion of 
 the biological mechanism of cell uptake and drug release from the drug carrying 
 device . in conclusion ,
the synthesis 
 and characterisation of a new example of a polymeric drug delivery system 
 derived from aspartic acid and lysine is presented .
many 
 potentially good pharmaceutical agents are either dose limited , or excluded 
 from use in clinical therapy due to insolubility in an aqueous medium , or 
 because of the many severe side effects that they may exhibit .
as far as cisplatin , the most successful 
 metal - containing chemotherapeutic drug of recent times , is concerned , these 
 side effects include inter alia : 
 poor aqueous solubility;intensive damage to the linings of the intestines , which leads to the loss of appetite ( anorexia ) and eventually 
 starving in the case of mice and rats ; severe 
 nausea , vomiting , and audio toxicity ; high 
 toxicity towards the kidneys and bone marrow ; being a 
 moderate carcinogen itself .
it can 
 induce lung cancer , skin papillomas , and other carcinomas . 
 in general ,
most if not all chemotherapeutic drug side effects are due to their 
 general inability to differentiate between diseased and healthy cells .
poor aqueous solubility ; intensive damage to the linings of the intestines , which leads to the loss of appetite ( anorexia ) and eventually 
 starving in the case of mice and rats ;   severe 
 nausea , vomiting , and audio toxicity ;   high 
 toxicity towards the kidneys and bone marrow ;   being a 
 moderate carcinogen itself .
even if a synthetic chemotherapeutic drug has few side effects , it does not imply 
 success in clinical use .
one must 
 realize that chemotherapeutic agents usually are poisons , or at the very least , 
 they are foreign to the body .
the 
 defence mechanism of the body , the reticuloendothelial system , recognizes them 
 as such and tries to remove them as fast as possible .
cisplatin , for example , is eliminated from the body in a biphasic 
 excretion mechanism in such a way that 50% of the initial administered dose is 
 excreted within 20 hours .
after 110 
 hours , 70% of the initial administered amount of drug is removed from the body 
 by the reticuloendothelial system .
the 
 principle mode of excretion in mice appears primarily to be through the urine , 
 90% of the injected dose being excreted within five days after injection .
this quick excretion rate causes large drug 
 concentration fluctuations in the body , and hence , also unpredictable 
 therapeutic activity , a highly undesirable situation .
the negative impact of 
 such drug concentration fluctuations in the body becomes apparent when one 
 relates it to 50% lethal dosages ( ld50 values ) , optimum dosages , 
 and a dosage having no influence on the diseased site at all .
for cisplatin , the ld50 dosage applicable to mice is 14  mg / kg mass of test animal , the optimum dose 
 is half of this , 7  mg / kg test animal , but 3  mg / kg mass of the test animal 
 has absolutely no influence on the diseased site at all . with the excretion profile that cisplatin 
 shows ,
it is clear that it is extraordinary difficult to maintain drug levels 
 in a patient close to the optimum dose level , but still below the ld50 value for prolonged times .
the initial 
 required overdose of drug that is required to maintain useful drug levels in 
 the body explains much 
 of the negative side effects of chemotherapeutic drugs .
a 
 further point of consideration in developing and administering anticancer drugs 
 is the development of drug resistance by a tumour after prolonged use .
a neoplastic population of cells is not a 
 static entity , but rather an ever - changing one .
tumours display an amazing ability to escape 
 or neutralize the actions of chemotherapeutic agents to which they were 
 initially sensitive .
some common 
 examples of this evasive ability are as follows : 

 the loss of specific receptors;down - regulation 
 of tumour - associated antigens ; andshedding of 
 antigens into the body fluids . 

the metastatic nature of cancer cells thus leads to the development of drug 
 resistance after continued drug dosage [ 11 , 12 ] . the loss of specific receptors ; down - regulation 
 of tumour - associated antigens ; and shedding of 
 antigens into the body fluids .
one 
 of the most fundamental barriers to selective drug delivery involves the 
 so - called targeting problem .
ideally , one would like a toxic agent that 
 can discriminate between neoplastic and nonneoplastic cells .
this approach presumes the existence of 
 something to aim at , that is , some molecular characteristic that differs 
 between target and nontarget cells .
even if a target can be identified , or a cell 
 need can be exploited , as has been done in 
 vitro , the problem remains of actually bringing the pharmaceutical 
 agent , in vivo , from the point of administering to the neoplastic 
 growth . to achieve this ,
many 
 physiological barriers including the reticuloendothelial system of the body 
 have to be bypassed .
consequently , 
 to improve the treatment of cancer , research today focusses on developing new 
 and better chemotherapeutic agents having less side effects ( i.e. , drugs that can zoom onto diseased 
 sites ) , new methods of drug delivery are being researched , combination 
 therapy is being investigated in the hope of finding synergistic effects , 
 the scope of radiation therapy is broadened , and completely new methods of 
 combating cancer are sought .
new 
 metal - based drugs include members of the titanocene and ferrocene family of 
 compounds [ 13 , 15 ] . a promising example 
 of a new method of cancer treatment focusses on photodynamic therapy ( pdt ) [ 16 , 17 ] . in pdt ,
the photo - active drugs in themselves 
 are inactive towards both healthy and cancer cells . however ,
when light of the correct 
 wavelength is shone on them , the drugs become photo - activated and give rise to conditions 
 that kill the cells 
 it is in contact 
 with , normally via the generation of singlet oxygen .
a good measure of selective cancer cell 
 killing is therefore induced into the treatment of a patient merely by 
 focussing light from a suitable laser source onto a tumourous growth .
healthy cells will stay largely undamaged 
 provided that light 
 is not shone upon it .
if cancer cells 
 would selectively and preferentially absorb the pdt drug , this would lead to a 
 further enhancement of selective cancer cell destruction .
some of them were found to be distributed upto 
 90% selectively in cancer cells , and only 10% in healthy cells after a certain 
 induction period .
many detrimental properties and side effects of promising anticancer drugs may at 
 least be partially overcome if the pharmaceutical agent is covalently anchored 
 to a polymeric drug carrier .
polymeric 
 or macromolecular drug - carrying devices should be distinguished from sustained drug - release 
 systems .
sustained drug - release systems 
 are normally associated with polymers ; frequently insoluble ; and in the form of 
 powders , pellets , and capsules from which the slow release of an encapsulated 
 drug from the  container  interior is possible . they normally do not represent systems that 
 may affect target - specific drug 
 delivery as much as they are devices for the continued release of drugs .
the term  polymeric drug carrier  describes 
 a polymer or macromolecule that contains covalent binding sites that are available to anchor active pharmaceutical 
 units as terminal or pendent groups protruding from the side of the polymeric 
 chain .
the naturally occurring ones such as antibodies [ 19 , 20 ] induce a high 
 mode of selective drug action onto the polymeric drug - containing device , but 
 usually suffer from a lack of large amounts of drug - anchoring sites .
synthetic polymers on the other hand are 
 more prone to suffer from immunogenic side effects , but this may be 
 overcome by copolymerisation with ethylene glycol fragments .
their big advantage is that they can be tailor - made 
 to be suitable for almost any purposes and can be engineered to have a large 
 amount of drug - anchoring sites .
the 
 clinical administration of a polymer bound drug , as compared to the free agent , 
 may significantly enhance the therapeutic effectiveness in terms of the 
 following : 
 accelerated 
 and unencumbered distribution in the aqueous central circulation system of the 
 body ( the blood ) , thereby reducing the risks of premature degradation and 
 excretion ; 
 they facilitate cell entry via 
 endocytosis  a cell membrane penetration mechanism 
 generally unavailable to nonpolymeric compounds but highly desired for drugs 
 operating intracellularly ; 
 more 
 precisely controlled drug serum levels ( i.e. , the restriction of the drug 
 concentration to the gap between toxic and minimum effective levels ) ; 
 an enhanced 
 depot effect through slow drug release from the polymer - drug conjugate . 

the 
 structural features of the polymer - drug conjugate required to comply with these 
 attributes include the following : 

 a highly 
 flexible linear main chain comprising structural entities that can provide 
 water solubility;a sufficiently large molecular mass to prevent quick excretion from the body ( the 
 threshold for elimination via the 
 kidneys is ca .
70  000  g mol1);a 
 biodegradable carrier backbone prone to catabolic elimination of the  spent  
 polymer main chain after the payload of drug has been delivered to the target 
 site ; 
 a large 
 amount of reactive functional groups as suitable binding sites for drug 
 attachment , these sites should be 
 distanced from the main chain by short ( 515 constituent atoms ) spacer segments 
 to diminish the steric bulk effect of the polymeric carrier backbone , and they 
 should be sufficiently separated from each other along the backbone to prevent 
 intramolecular multifunctional drug binding;the 
 separation of binding sites on the polymeric backbone requires insertion of 
 side chains on the main chain between active binding sites that lack drug - binding 
 capabilities , but enhance other desirable properties .
these may include enhancing the hydrophilic 
 nature of the ultimate polymeric drug - carrying device , the incorporation of 
 moieties that enhance drug - targeting capability ( i.e. , a tumour homing device ) , 
 antispasmodic properties , or any other desirable physiological effect ; 
 one or more 
 biofissionable functions ( amide , ester , urethane , disulphide , etc . ) to be 
 inserted into the carrier - drug connecting link .
at least one of these must be sufficiently 
 remote from the main chain to permit enzyme approach and cleavage action to 
 result in effective drug release in a targeted biological compartment ; 
 carrier 
 backbones and spacers which are non toxic per se .
they should 
 especially possess absolute minimal immunogenicity so as to preclude 
 carrier - induced pathological effects . 

akey component towards the success of polymeric drug - carrying devices centres 
 around biodegradable bonds and their capability to home preferentially onto a neoplastic 
 growth .
apart from targeting fleeting 
 receptors on a collection of metastatic neoplastic cells , the nutritional needs 
 of such cells may be utilized to gain preferential neoplastic cell access .
neoplastic cells grow much faster than 
 healthy cells , and have a much higher demand for food in the form of amino 
 acids and energy .
hence copolymers 
 constructed at least partially from amino acids are very attractive as 
 polymeric drug delivery devices .
sugar groups as a 
 source of energy will especially enhance preferential uptake by neoplastic 
 cells .
other biodegradable groups that 
 may be employed include disulphide bonds that may be reduced to thiol groups 
 , or , since cell interior has a lower ph than blood plasma , for example , 
 weakly acid - labile groups such as thio - ethers . accelerated 
 and unencumbered distribution in the aqueous central circulation system of the 
 body ( the blood ) , thereby reducing the risks of premature degradation and 
 excretion ; 

they facilitate cell entry via 
 endocytosis  a cell membrane penetration mechanism 
 generally unavailable to nonpolymeric compounds but highly desired for drugs 
 operating intracellularly ; 
 more 
 precisely controlled drug serum levels ( i.e. , the restriction of the drug 
 concentration to the gap between toxic and minimum effective levels ) ; 
 an enhanced 
 depot effect through slow drug release from the polymer - drug conjugate . 
 a highly 
 flexible linear main chain comprising structural entities that can provide 
 water solubility ;
a sufficiently large molecular mass to prevent quick excretion from the body ( the 
 threshold for elimination via the 
 kidneys is ca .
g mol1 ) ; a 
 biodegradable carrier backbone prone to catabolic elimination of the  spent  
 polymer main chain after the payload of drug has been delivered to the target 
 site ; 
 a large 
 amount of reactive functional groups as suitable binding sites for drug 
 attachment , these sites should be 
 distanced from the main chain by short ( 515 constituent atoms ) spacer segments 
 to diminish the steric bulk effect of the polymeric carrier backbone , and they 
 should be sufficiently separated from each other along the backbone to prevent 
 intramolecular multifunctional drug binding ; the 
 separation of binding sites on the polymeric backbone requires insertion of 
 side chains on the main chain between active binding sites that lack drug - binding 
 capabilities , but enhance other desirable properties .
these may include enhancing the hydrophilic 
 nature of the ultimate polymeric drug - carrying device , the incorporation of 
 moieties that enhance drug - targeting capability ( i.e. , a tumour homing device ) , 
 antispasmodic properties , or any other desirable physiological effect ; 
 one or more 
 biofissionable functions ( amide , ester , urethane , disulphide , etc . ) to be 
 inserted into the carrier - drug connecting link .
at least one of these must be sufficiently 
 remote from the main chain to permit enzyme approach and cleavage action to 
 result in effective drug release in a targeted biological compartment ; 
 carrier 
 backbones and spacers which are non toxic per se .
they should 
 especially possess absolute minimal immunogenicity so as to preclude 
 carrier - induced pathological effects . 

although 
 models as those mentioned 
 above for synthetic polymeric drug carriers have been in existence for some time , until recently only a few efforts were made to actually prepare 
 such compounds .
a review by neuse   describes some of the most recent researches in this 
 field .
scheme 1 illustrates the 
 synthetic approach to actually obtain such polymeric drug - delivering 
 devices .
the centre of scheme 1 serves 
 to visualise the overall strategy . at 
 the left and right ends of scheme 1 an example for an actual synthesis of a polymeric 
 drug delivery system  
the use of the water - soluble 
 polyaspartic acid drug carrier not only allowed the water - insoluble parent 
 ferrocene - containing drug to be well soluble in water in concentrations 
 exceeding 100  g dm3 but it also increased the cytotoxicity of the active drug as 
 tested on murine emt-6 cancer cells from an ld90 value of 500  g / cm3 in the case of free 
 3-ferrocenylbutanoic acid to 80  g / cm3 drug content for the polymeric 
 drug delivery device . at 
 least two factors probably contribute to the enhanced activity of the above - mentioned polymeric 
 drug delivery device .
the first must be 
 related to the enhanced aqueous solubility of the ferrocene drug .
free ferrocene itself , which is insoluble in 
 water , has been shown to be completely inactive as cytotoxic agent .
the second factor centres around the use of 
 aspartic acid as monomeric subunit in the main chain of the polymeric drug carrier . as the cancer cells have a large need for 
 nutrients such as amino acids , the use of a polyamino acid as drug carrier may 
 cause the drug carrying device to be absorbed faster and more efficiently by 
 cancer cells than would be the case for the free drug .
a key factor should be observed in the synthetic sequence shown 
 in scheme 1 .
the use of 
 tri- or higher - functional polymerization monomers is useful in the synthesis of 
 polymeric drug carriers , as it provides a free arm where the drug may 
 eventually be anchored .
only two of the 
 three or more monomer functionalities should be used to obtain a linear , highly 
 flexible , and soluble monomer .
however , normally the use of tri- or higher - functionalised 
 monomers will lead to the formation of highly crosslinked , rigid , and 
 insoluble polymers . in the case of 
 aspartic acid
, this did not happen because the formation of stable 
 five - membered succinimide rings is thermodynamically highly favoured .
it effectively masked the second carboxylic 
 acid functional group of aspartic acid from unwanted crosslinking 
 reactions .
for other tri- or higher - functionalized 
 amino acids , such as trifunctionalised lysine or tetrafunctionalised cystine 
 , protection of the additional functional groups is required to prevent 
 formation of pharmaceutically undesired crosslinked polymers .
uptake by neoplastic ( and healthy ) cells 
 of the soluble polymeric device , shown in scheme 1 , probably occurs by 
 endocytosis , in particular , by fluid - phase pinocytosis as described by duncan 
 and kopeek .
as shown in figure 1 , 
 polymers in solution are internalised by means of fluid - phase pinocytosis .
cell internalisation may also occur if the 
 polymeric drug delivery device binds to surface cell receptors by means of 
 adsorptive pinocytosis .
polymeric drug carriers with porphyrins or 
 phthalocyanines attached to them will probably gain access to neoplastic cells 
 by means of adsorptive pinocytosis as this would be consistent with the 
 selective uptake of these macrocycles by cancer cells .
it was 
 found that pinocytotic uptake by cells of polymers bearing phenol - containing 
 side chains was greatly enhanced . 

once internalized , fusion of the pinocytotic vesicles with primary lysosomes 
 takes place to form secondary lysosomes . 

release of the drug from the polymeric drug carrier inside the secondary 
 lysosomes occurs by enzymatic hydrolysis of the biodegradable amide bonds that 
 link the drug , ferrocene in scheme 1 , with the polymeric drug carrier , a 
 polyaspartic acid derivative in scheme 2 . 

primary lysosomes may contain more than 50 hydrolytic enzymes that are 
 generated in the golgi apparatus .
next , 
 the drug and other useful low - molecular - mass degradation products like amino acids 
 are released from the secondary lysosomes for use inside the cell nucleous .
finally , the  spent  polymer main chain and 
 any other nondegradable material leave the cell by exocytosis .
no drug is released outside a living cell because these drugs are not in 
 contact with appropriate hydrolytic enzymes . 
 as the folding pattern of the polymeric drug carrier normally 
 inactivates the drug while polymer - bound , it means no negative side effects can 
 be induced by the drug prior to release from the polymeric main chain .
in addition , if the polymeric drug delivery 
 device could be designed in such a way that it is internalized selectively into 
 only neoplastic cells , the negative side effects associated with chemotherapy 
 could be circumvented completely .
lysine , 1 , introduces a new dimension into 
 our discussion of the design of polymeric drug carriers . to prevent crosslinking reactions ,
one can either protect the - or the -amino group , scheme 2 , to obtain lysine 
 derivatives 3 or 4 , respectively .
only -amino trifluoroacetyl protected lysine , 3 , is obtained by direct treatment of 
 lysine with trifluoroacetic anhydride in trifluoroacetic acid because the -amino group is much more basic than the -amino group .
the 
 zwitter - ionic structure of lysine , 1 , 
 implies that the -amino 
 group is mainly in the protonated form and thus well protected against 
 acylation reactions .
however , treatment 
 of 1 with ethyl 
 thioltrifluoroactetate under basic conditions   according to the method 
 described by schallenberg gave n - trifluoroacetyllysine , 4 , in 33% yield after 
 recrystallisation from hot water / ethanol in a 2 : 3 ratio .
it is advantageous to use the -amino group rather than the -amino group eventually as the drug - anchoring 
 site because it separates it far enough from the polymer main chain to allow 
 easy enzyme approach to initiate cleavage of amide bonds to facilitate drug 
 release from the eventual drug - carrying devices 1113 .
if the -amino group is protected for later drug - anchoring 
 purposes , the steric bulk effect of the polymeric main chain may be so large 
 that enzyme - induced drug release will become kinetically very slow .
in addition , it was also previously 
 demonstrated   that drug - anchoring reactions of the type shown in scheme 2 
 to generate 1113 via amide bond formation proceed more effectively with compounds 
 possessing longer side chains .
we 
 describe here the syntheses , from n - trifluoroacetyllysine 4 , aspartic acid 5 , and polymeric precursors 6 and 7 of polymeric drug carriers 8 and 9 , scheme 2 .
we also 
 describe how the ferrocenyl and phthalocyanine moieties may be covalently 
 anchored to the carrier polymers 8 and 9 utilising the coupling agent 10 to obtain the polymeric drug 
 delivery devices 11 , 12 , and 13 .
it 
 was not very easy to predetermine reaction conditions to obtain a specific 
 target x / y ratio in polymer 6 because of a tendency towards homopolymerisation by aspartic acid . in first experiments , 4 and 5
were thermally 
 copolymerised in a monomer mole ratio of 1 : 10 ( 4 : 5 ) at 180c under nitrogen and atmospheric pressure in 
 the presence of a polyphosphoric acid ( ppa ) mass fraction of 0.8 for 8 hours .
the first was extracted with water , and 
 after dialysis in an 
 8,000 molecular mass cutoff membrane tubing , 6 was isolated having an x / y = 2.6/1 ratio , and inherent viscosity inh = 0.06  dl g in 3% 
 yield , m.p = 301c ( dec . ) . 

the x / y ratio was established by comparing the h nmr signal 
 integrals at 5.15.5  ppm ( ch of imide , one proton per closed imide unit ) , 
 4.44.6  ppm ( ch of aspartic acid , the integral values showed only 65% ring 
 closure of the aspartic acid recurring unit occurred ) , with that of the 
 combined  ,  , and  methylene protons of the lysine recurring unit 
 ( 6 protons per lysine recurring unit ) at 0.91.8  ppm .
the remaining water - insoluble residue was 
 dissolved in dmf and precipitated with ethanol to yield 56% polymer having inh(dmf ) = 0.07  dl g , 
 m.p = 286c ( dec . ) in this case , the x / y unit ratio was 
 established by h nmr as 22/1 . 

the remainder of material had molecular mass 
 < 8,000  g mol1 , and was dialysed away .
when 4 and 5 were thermally co - polymerised in a monomer mole ratio of 1 : 6 = 4 : 5 , 
 other conditions still being the same , the water - soluble fraction was isolated 
 in 8% possessing a monomer ratio of x / y = 1.4/1 .
the dmf - soluble fraction was isolated in 30% yield having a monomer ratio of x / y = 25/1 . in another experiment , when the 
 ppa mass fraction was lowered from 0.8 to 0.5 , the water - soluble fraction was very 
 small , but the dmf - soluble fraction was isolated in 50% yield having also a 
 monomer ratio of x / y = 25/1 .
the 
 above results showed that 4 and 5 have a tendency to 
 homopolymerise .
the low lysine content 
 in each recovered fraction was attributed to the tendency of -amino acids to form 6-membered 
 diketopiperazine rings upon heating . 
 in the case of 4
, this would 
 result in termination of any polymerisation process , and account also for the 
 large % of low - molecular - mass material that was lost upon dialyses .
c nmr spectroscopy showed that 
 the trifluoroacetyl protective group did not survive the polymerisation 
 conditions .
however , loss of the 
 trifluoroacetyl protective group must have happened in the latter stages of 
 polymerisation , since no insoluble portion was found in the workup of 6 . 

crosslinking was , therefore , ruled out as the cause of the relatively 
 moderate yields .
further 
 experiments focussed exclusively on a 1 : 2 = 4 : 5 monomer mole ratio 
 in a ppa mass fraction of 0.5 at 180c for only 2.5 hours at 
 pressures below 2  torr .
these 
 conditions resulted in almost no water - soluble fraction ( < 1% ) , and a 
 dmf - soluble fraction ( 18% yield , decomposition temperature = 269c ) 
 having a repeating unit ratio of x / y = 7/1 and 29% of aspartic acid fragments 
 still
c nmr 
 showed that the trifluoroacetyl protective group remained intact when using 
 these shorter reaction times . by 
 substituting ppa with 85% ortho 
 h3po4 , repeated 
 experiments gave products having recurring unit ratios ranging 2/1 < x / y 

the increase in yield and 
 better recurring unit ratios probably arose because it is easier to mix 4 and 5 to a homogeneous distribution in ortho h3po4 than in ppa .
lower reaction 
 temperatures ( 140c ) lowered yields substantially to only 6% and 
 virtually the entire product became water - soluble , even after 7 hours of 
 reaction time .
the 
 absence of any h nmr signals at 5.25  ppm , as well as the presence 
 of the signal at 4.24.6  ppm showed that for this product , ring closure of the 
 aspartic acid fragment did not occur .
it had a repeating unit ratio of x / y = 
 4/1 as compared to the target of 2/1 .
c 
 nmr showed that the trifluoroacetyl protective group remained intact under 
 these conditions of decreased temperatures even though the reaction time was 
 increased to 7 hours .
nucleophilic 
 attack of amines on the succinimide rings of polymeric precursor 6 takes place with ease , and the 
 success rate is independent of x / y ratios . 
 by choosing the correct nucleophiles
, the solubility properties of 
 choice may be introduced to the carrier polymer . here ,
the 3-aminopropyl 
 morpholine unit was used to generate water - soluble 8 in 83% yield after stirring the intermediate product 7 with 2% naoh solution to remove the 
 trifluoroacetyl protecting group and dialysis in 8,000 molecular mass cutoff 
 membrane tubing .
although intermediate 7 can be isolated en route to 8 , this is not necessary .
2-phenylethyl amine was used to generate white , water - insoluble 9 in 90% yield after removal of the 
 protective group and slow precipitation with water from a dmf solution .
to 
 demonstrate the carrier capabilities of 8 and 9 , the antineoplastic ferrocenyl 
 moiety   and an example of the family of drugs that may be used in 
 photodynamic cancer therapy , a phthalocyanine , have been anchored on these 
 polymeric drug carriers .
both ferrocene and phthalocyanine are insoluble in 
 water when not functionalised , or as carboxylic acid derivatives . even as 
 sodium carboxylates , they are poorly soluble in water . however , once anchored on the polymeric drug 
 carrier 8 , the ferrocene - containing 
 derivative 11 was soluble in excess 
 of 30  g / dm3 , while the phthalocyanine derivative exceeded an aqueous 
 solubility of ca
. 5  g dm3 at ph s more extreme than those found in 
 the gastrointestinal system ( 18.5 ) [ 32 , 33 ] . 

however , prior to drug anchoring , the ferrocene and phthalocyanine 
 fragments needed to be modified in such a way that coupling with the available amine 
 active sites on the polymeric drug carriers 8 and 9 is possible . to 
 generate a biodegradable bond between drug and polymeric carrier , formation of 
 a peptide ( i.e. , amide ) , saccharide , or nucleotide bonds  
the amide bond was aimed for as a 
 biodegradable bond linking carrier to drug . 

thus , 3-ferrocenylbutanoic acid   and cobaltotetracarboxyphthalocyanine 
  were prepared according to known procedures for later reaction with the 
 amine - containing polymeric carrier devices 8 and 9 .
although 
 polymers 8 and 9 , the ferrocene - containing derivatives 11 and 13 , and the 
 phthalocyanine - containing derivative 12 are robust , frequently the drug moiety of interest ( as opposed to the drug 
 carrier ) may be very labile .
it is advantageous , 
 therefore , to be able to anchor the drug onto the polymeric carrying device 
 under very mild conditions in order to preserve labile moieties .
also , coupling procedures need to be 
 developed that will allow coupling in media in which both the water - soluble 
 drug carrier , here 8 and 9 , and poorly soluble or 
 aqueous - insoluble drug may be partially dissolved or suspended . coupling with the aid of 
 o - benzotriazolyl - n , n
, n,n-tetramethyluronium 
 hexafluorophosphate , 10 , at room temperaturein dmf or dmso , sometimes with thf or 
 water as cosolvent , satisfies this requirement .
24-hour reactions between 
 tetracarboxyphthalocyanine and 8 with a x / y ratio of 3/1 in the presence of this coupling agent led to only ca . 

10% phthalocyanine binding in 12 as 
 ascertained by h nmr utilising the phthalocyanine phenyl aromatic 
 proton signal ( 12 hours per phthalocyanine group ) at ca .
the 
 closeness of the cooh functional group in the phthalocyanine moiety had two 
 effects , both associated with an extremely slow coupling rate due to the 
 closeness of the bulky phthalocyanine core ( slow coupling reactions because of 
 steric crowding have already been commented on , and are described in reference 
 25 ) .
firstly , it explains the low 
 binding efficiency ( z=0.1y ) of cobaltotetracarboxyphthalocyanine to polymeric 
 drug carrier 8 .
secondly , it explains why crosslinking 
 reactions involving cobaltotetracarboxyphthalocyanine apparently did not take 
 place to a noticeable extent .
as 
 ascertained by h nmr , no precipitate from the dialysis tubing other 
 than free cobaltotetracarboxyphthalocyanine ( i.e. , unbound ) could be found . in 
 contrast
, 3-ferrocenylbutanoic acid was anchored to a derivative of 8 having a x / y ratio of 2/1 ca .
7080% effectively ( i.e. , zaverage=0.75y ) within 1 hour at room 
 temperature to give 11 as 
 product .
this result was obtained by 
 comparing h nmr signals ( obtained in d2o ) of the ch2o 
ch2 morpholine signals ( 4 hours per morpholine group ) at 3.53  ppm with those of the 
 ch3 substituent in 3-ferrocenylbutanoic acid at 1.1  ppm ( it is a 
 well - defined peak that can be uniquely identified next to the lysine signals ) , 
 the combined  ,  , and  methylene protons of the lysine recurring unit 
 ( 6 protons per lysine recurring unit ) at 1.21.6  ppm , and the aspartate ch 
 protons at 4.24.5  ppm .
an atomic 
 absorption iron elemental analysis for 11 with x / y = 2/1 showed an iron content of 4.8% which corresponds to z=0.67y . 
 
coupling 
 of 3-ferrocenylbutanoic acid to a derivative of 9 with x / y ratio 3/1 to obtain 13 in dmf as solvent was more successful , probably because there was no solvent 
 water present during the coupling reaction , see experimental . by comparing the integral values of the 
 phenyl group h nmr signals ( in dmso - d6x ) at 7.15  ppm 
 with those of the ch3 substituent in 3-ferrocenylbutanoic acid at 
 1.2  ppm ( it is a well - defined peak that can be uniquely identified between the 
 lysine signals ) and the combined  ,  , and  methylene protons of the lysine recurring unit 
 ( 6 protons per lysine recurring unit ) at 0.91.6  ppm
, it was ascertained that 
 the ferrocenyl group was anchored 94% efficiently , that is , z=0.94y .
an atomic absorption iron elemental analysis 
 for 13 with x / y = 3/1 showed an iron 
 content of 5.08% which corresponds to z=0.87y .
the 
 water - insoluble polymer 12 is of 
 particular interest because it demonstrates two separate functional moieties 
 can be supported on the same polymer drug carrier , here phenyl and 
 ferrocenyl .
this begs the 
 question :  can more than one 
 antineoplastic group be anchored on the same polymeric drug carrier ?  if this would be possible , combination 
 therapy would be possible without administering more than one type of drug in 
 separate doses to a patient .
the synergistic effect usually associated with 
 combination therapy [ 36 , 37 ] would then be potentially obtainable in a single 
 drug dose by binding to the carrier polymer all the drugs required .
biological studies are at 
 the moment in progress to determine if present lysine - containing polymeric drug - carrying 
 devices 11 and 12 can compare in efficiency with previously described polyaspartic 
 acid drug - delivering systems .
chemicals were from merck & co. , inc . ( nj , 
 usa ) 
 or sigma - aldrich and used without further 
 purification .
nmr measurements , at 298  k , were recorded on a bruker advance dpx 300 nmr spectrometer .
chemical shifts are reported as  values 
 relative to sime4 at 0  ppm .
infrared spectra were recorded on a hitachi 
  27050 infrared spectrometer in a kbr 
 matrix .
viscosity measurements were 
 made in a cannon - fenske tube at 35.2c in water or dmf .
iron 
 analyses were performed on a varian techtron atomic absorption spectrometer at 
 272  nm .
n - trifluoroacetyllysine , 4 ,   and cobalto 
 tetracarboxyphthalocynanine   were prepared as described before .
a representative example of 
 the synthesis of polymer 6 : aspartic acid ( 1.331  g ; 10  mmol ) , n - trifluoroacetyllysine ( 1.211  g ; 5  mmol ) , 
 and polyphosphoric acid ( 2.542  g ) were thoroughly mixed 
 in a 250  cm round - bottom flask .
the flask was 
 mounted on a rotating evaporator , and lowered ( while rotating slowly ) into an 
 oil bath preheated to 180  . 
 the pressure was reduced after 5 minutes to below 2 torr with the aid of 
 a mechanical pump . after 2.5 hours of 
 slow rotation
dialyses of the water 
 extract for 16 hours gave after freeze drying only a negligible quantity of 
 product .
precipitation by ethanol afforded 0.38 g ( 18% ) of 6 ; inh(dmf ) = 0.08  dl g , 
 x / y = 7/1 ( see discussion text for conditions to obtain other x / y ratios ) ; m.p . 
 295  c ( dec . ) .
h 
 nmr ( dmso - d6-x ) : 0.91.8 ( 6h ; 
  ,  and  ch2 s of lysine ) ; 2.1 ( 3.8 h ; ch2 of open aspartic acid fragment ) ; 2.62.9 
 ( 4.8 h ; ch of succinimide ) ; 3.03.4 ( 4.8 h ; ch of succinimide ) ; 4.44.6 ( 1.9 h ; ch of aspartic acid 
 fragment ) ; 5.15.5 ( 4.8 h ; ch of succinimide ) .
c nmr ( dmso - d6-x ) : 110122 , q , cf3 ; 
 155 - 156 , q , cocf3 . 

ir : 1798 , 1720 ( c = o ) ; 1219 , 1189 
 ( cf3 ) .
polymers 7 , and 9 : a solution of 0.21 g [ 0.5  mmol , 
 inh = 0.08  dl g , x / y = 
 3/1 ] of 6 and 2-phenylethyl amine 
 ( 0.202  g ; 2  mmol ) in 5  cm dmf was stirred for 5 hours at ambient 
 temperature .
the newly formed 7 was not isolated ( although it can be 
 by precipitation with ethanol or water ) , but stirred for 5 hours with 20  cm of a 2% naoh solution .
the resulting 
 clear reaction mixture was dialysed for 16 hours , and freeze dried to give 0.24  g ( 90% yield ) of 9 as a white solid 
 which would not redissolve in pure water , but was soluble in dmso and dmf ; inh(dmf ) = 0.07  dl g ; 
 x / y = 3 ; m.p 275c ( dec . ) . polymer 8 : this polymer was obtained 
 in exactly the same way as polymer 9 , 
 but by replacing 2-phenylethyl amine with 3-aminopropyl morpholine ( 0.289  g ; 2  mmol ) , and the x / y ratio of 6 was 
 2/1 .
characterisation data for 8 : yield = 83% ; inh = 0.06  dl g ; x / y = 
 2 ; m.p .
196c ( dec . ) . 
 unlike 9 , this polymer 
 remained water - soluble after dialysis and freeze drying .
polymer 11 : to a solution of 0.2  g 
 ( 0.24  mmol ; inh = 0.06  dl g ; x / y = 2 ) of 8 in 1  cm water was added ( in the correct order ) 0.035  g ( 0.35  mmol ) 
 triethylamine , a solution of 0.068  g ( 0.25  mmol ) 3-ferrocenylbutanoic acid in 1  cm thf ( the thf may be replaced with dmf ) and 0.114  g ( 0.3  mmol ) of 
 o - benzotriazolyl - n , n , n,n-tetramethyluronium 
 hexafluorophosphate , 10 .
the mixture was stirred for one hour at room 
 temperature during which time the heterogeneous mixture homogenized .
water ( 10  cm ) was added to the 
 reaction mixture before it was centrifuged , dialysed for 16 hours in an 8,000 
 molecular mas cutoff membrane tubing and freeze dried to give 0.11  g ( 44% ) of 11 ; inh = 0.04  dl g ; x : y : z = 2.00 : 0.35 : 0.65 ; m.p .
h nmr ( d2o ) : 
 1.01.2 ( 0.65 of 3h , ch3 ) ; 1.21.6 ( 6h ;  ,  and  ch2 s of lysine ) ; 3.53 ( 8h , ch2o  ch2 of morpholine ) , 3.94.2 ( 0.65 of 9h , c10h9fe ) ; 4.24.6 
 ( 3h , ch of aspartate ) .
ir / cm : 
 1660 ( c = o ) ; 1550 ( nh ) .
polymer 12 : the same procedure was used as for 11 except that the solvents were all dmf , 3-ferrocenylbutanoic acid 
 was replaced with an equivalent amount of cobalto tetracarboxyphthalocyanine 
 and the reaction time was increased to 24 hours . characterisation data : 44% yield ; inh = 0.04  dl g ; x : y : z 
 = 2.0 : 0.9 : 0.1 ; m.p . 194c 
 ( dec . ) ; h
nmr ( d2o ) : 1.21.6 ( 6h ;  ,  and  ch2 s of lysine ) ; 3.53 ( 8h , ch2o  ch2 of morpholine ) ; 4.24.6 ( 3h , ch of aspartate ) 7.38.3 ( 0.1 of 12h , phthalocyanine 
 aromatics ) ; ir / cm : 1720 , 1660 ( c = o ) ; 1550 ( nh ) .
polymer 13 : the same procedure was used as for 12 , except that 8 was 
 replaced by an equivalent amount of 9 , tetracarboxyphthalocyanine was 
 replaced with a solution of 0.068  g ( 0.25  mmol ) 3-ferrocenylbutanoic acid 
 dissolved in 1  cm dmf and the reaction was allowed to proceed for 1 
 hour only .
characterisation data : 64% 
 yield ; inh = 0.04  dl g ; x : y : z = 3.00 : 0.13 : 0.87 ;  m.p .
h nmr ( dmso - d6-x ) : 1.2 
 ( 0.87 of 3h , ch3 ) ; 0.91.6 ( 6h ;  ,  and  ch2 s of lysine ) ; 4.1 - 4.2 ( 0.87 of 
 9h , c10h9fe ) ; 4.24.6 ( 4h , ch of aspartate ) .
ir / cm : 1655 ( c = o ) ; 1553 ( nh ) .
in summary , it was shown that upon using the thermal polymerisation technique , 
 aspartic acid and n - trifluoroacetyllysine 
 both have a tendency to homopolymerise . 

this tendency becomes stronger as reaction conditions become harsher , 
 such as higher temperatures , longer reaction times , or lowering of pressures .
ring closure of the aspartic acid fragment becomes progressively less 
 efficient when milder reaction conditions are employed .
it was also shown that the trifluoroacetyl 
 protective group is less stable at elevated temperatures for prolonged periods 
 of time .
chemical removal of the 
 trifluoroacetyl protective group was achieved under mild alkaline conditions to 
 liberate free amino groups on side chains of lysine - containing potential 
 polymeric drug carriers .
coupling of a 
 carboxylic acid - functionalised ferrocene with the amine - containing polymeric 
 drug carrier was effectively achieved utilising o - benzotriazolyl - n , n , n,n-tetramethyluronium 
 hexafluorophosphate as coupling agent . 

this was 
 presumably due to the poor solubility of cobaltotetracarboxyphthalocyanine in 
 the required solvents .
 the general synthetic strategy towards water - soluble biodegradable drug carriers and the properties that they must have are discussed . 
 the syntheses of water - soluble biodegradable copolymers of lysine and aspartic acid as potential drug - delivering devices , having amine - functionalised side chains are then described . 
 covalent anchoring of carboxylic acid derivatives of the antineoplastic ferrocene and photodynamically active phthalocyanine moieties to the amine - containing drug carrier copolymers under mild coupling conditions has been achieved utilising the coupling reagent o - benzotriazolyl - n , n , n,n-tetramethyluronium hexafluorophosphate to promote formation of the biodegradable amide bond . 
 even though the parent antineoplastic ferrocene and phthalocyanine derivatives are themselves insoluble in water at ph 
 < 7 , the new carrier - drug conjugates that were obtained are well water - soluble . 
1. INTRODUCTION
2. PROBLEMS ASSOCIATED WITH CHEMOTHERAPEUTIC DRUGS
3. SYNTHETIC POLYMERIC DRUG DELIVERY SYSTEMS
4. SYNTHETIC STRATEGIES TOWARDS POLYMERIC DRUG DELIVERY SYSTEMS
5. MECHANISM OF CELL UPTAKE AND DRUG RELEASE
6. THE SYNTHESES OF LYSINE-BASED POLYMERIC DRUG CARRIERS
7. EXPERIMENTAL
8. CONCLUSIONS